Universal platform for car therapy targeting a novel antigenic signature of cancer

ABSTRACT

The present invention provides a method of identifying a target for preparing an inhibitory chimeric antigen receptor (iCAR) or a protective chimeric antigen receptor (pCAR) capable of preventing or attenuating undesired activation of an effector immune cell. Also provided are a list of iCAR targets, as well as vectors and transduced effector immune cells comprising the nucleic acid molecule and methods for treatment of cancer comprising administering the transduced effector immune cells are further provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/564,454, filed Sep. 28, 2017, and U.S. Provisional Application No.62/649,429, filed Mar. 28, 2018, each of which is herein incorporated byreference.

SEQUENCE LISTING

This patent application contains a Sequence Listing which has beensubmitted electronically in ASCII format and are hereby incorporatedherein by reference in its entirety. Said ASCII copy, created Sep. 27,2018, is named 120575-5003_ST25.txt.

ASCII TABLE

The provisional patent application to which the current applicationclaims priority contains a lengthy table section. A copy of the tablewas submitted to the U.S. Patent and Trademark Office on compact disc inASCII format with priority U.S. Provisional Application No. 62/649,429,filed Mar. 28, 2018 and is hereby incorporated by reference, and may beemployed in the practice of the invention. Said ASCII table, createdMar. 28, 2018, is as follows: 120575-5003-PRallCandExt1167Genes_5003_PR.txt, 272,719,870 bytes.

FIELD OF THE INVENTION

The invention relates to the field of cancer immunotherapy by adoptivecell transfer, employing activating chimeric antigen receptors (aCARs)recognizing antigens expressed on the surface of tumor cells, inhibitoryCARs (iCARs) and protective CARs (pCARs) directed at allelic variants ofthe same or other cell surface antigens expressed by normal cells butnot by the tumor due to loss of heterozygosity (LOH).

BACKGROUND OF THE INVENTION

The identification of targetable antigens that are exclusively expressedby tumor cells but not by healthy tissue is undoubtedly the majorchallenge in cancer immunotherapy today. Clinical evidence that T cellsare capable of eradicating tumor cells comes from numerous studiesevaluating highly diverse approaches for harnessing T cells to treatcancer (Rosenberg and Restifo, 2015). These approaches employ bonemarrow transplantation with donor lymphocyte infusion, adoptive transferof tumor-infiltrating lymphocytes (TILs), treatment with T cellsgenetically redirected at pre-selected antigens via CARs (Gross andEshhar, 2016a) or T cell receptors (TCRs), the use of immune checkpointinhibitors or active vaccination. Of these, the use of geneticallyengineered T cells and different strategies for active immunizationentail pre-existing information on candidate antigens which are likelyto exert a durable clinical response but minimal adverse effects. Yet,as stated in the title of a recent review by S. Rosenberg, “Findingsuitable targets is the major obstacle to cancer gene therapy”(Rosenberg, 2014).

The concept of using chimeric antigen receptors (or CARs) to geneticallyredirect T cells (or other killer cells of the immune system such asnatural killer (NK) cells and cytokine-induced killer cells) againstantigens of choice in an MHC-independent manner was first introduced byGross and Eshhar in the late 1980s (Gross et al., 1989). They areproduced synthetically from chimeric genes encoding an extracellularsingle-chain antibody variable fragment (scFv) fused through a flexiblehinge and transmembrane canonic motif to signaling components comprisingimmunoreceptor tyrosine-based activation motifs of CD3-ζ or FcRy chainscapable of T cell activation. At present, CARs are being examined indozens of clinical trials and have so far shown exceptionally highefficacy in B cell malignancies (Dotti et al., 2014; Gill and June,2015; Gross and Eshhar, 2016a). The safety of CAR-T cell therapy isdetermined, in large, by its ability to discriminate between the tumorand healthy tissue. A major risk and the direct cause for adverseautoimmune effects that have been reported in clinical and preclinicalstudies is off-tumor, on-target toxicity resulting from extra-tumorexpression of the target antigen (dealt with in detail in our recentreview (Gross and Eshhar, 2016b) and (Klebanoff et al., 2016)).Concerning this risk, shared, non-mutated cell surface antigens whichare currently tested clinically or pre-clinically for CAR therapy can begenerally divided into a number of categories according to their tissuedistribution and mode of expression:

-   -   Strictly tumor-specific antigens. Perhaps the only member in        this group which is already being examined clinically is variant        III of the epidermal growth factor receptor (EGFRvIII) that is        frequently overexpressed in glioblastoma and is also found in        non-small cell lung carcinoma and prostate, breast, head and        neck and ovarian cancers but not on normal tissue.    -   Surface antigens expressed on the tumor and on non-vital healthy        tissue. Potential CAR antigens in this group are        differentiation-related molecules that are mainly restricted to        the B cell lineage. Prominent among these (and a target antigen        in numerous clinical trials) is CD19, a pan-B cell marker        acquired very early in B cell differentiation and involved in        signal transduction by the B cell receptor (BCR). Membrane        prostate antigens constitute another class of antigens in this        category.    -   Antigens that are typically expressed by non-malignant        tumor-promoting cells. One such antigen is fibroblast activation        protein (FAP), a cell surface serine protease which is almost        invariably expressed by tumor-associated fibroblasts in diverse        primary and metastatic cancers. Another antigen is vascular        endothelial growth factor (VEGF), which is highly expressed        during tumor angiogenesis and is normally expressed on vascular        and lymphatic endothelial cells in many vital organs.    -   Tumor associated antigens (TAAs) shared with vital healthy        tissue.

Most other TAAs which are presently evaluated in preclinical andclinical studies are overexpressed by tumors but are also present,usually at lower level, on essential normal tissue.

The broad spectrum of strategies devised to tackle autoimmunity in CAR Tcell therapy can be divided into those which seek to eliminate, orsuppress transferred T cells once damage is already evident (reactivemeasures) and those that aim at preventing potential damage in the firstplace (proactive measures) (Gross and Eshhar, 2016a). Reactiveapproaches often use suicide genes such as herpes simplex virusthymidine kinase (HSV-tk) and iC9, a fusion polypeptide comprising atruncated human caspase 9 and a mutated FK506-binding protein. Otherapproaches utilize antibodies to selectively remove engineered cellswhich go havoc or, as recently demonstrated, a heterodimerizingsmall-molecule agent which governs the coupling of the CAR recognitionmoiety to the intracellular signaling domain (Wu et al., 2015). Whilesome proactive measures are designed to limit the in-vivo persistence orfunction of CAR T cells (for example, the use of mRNA electroporationfor gene delivery), others directly address the critical challenge ofincreasing antigenic selectivity of the therapeutic CARs so as to avoiddamage to non-tumor tissue. Two of these raise particular interest, asthey can potentially broaden the range of tumor antigens which can besafely targeted by CAR T cells:

-   -   Combinatorial (or ‘split’) antigen recognition. While true        tumor-specific surface antigens are rare, combinations of two        different antigens, not-necessarily classified as        tumor-associated antigens that are co-expressed by a given        tumor, can define a new tumor-specific signature. Restricting        the activity of CAR T cells to such antigen pairs provides a        critical safety gauge and, consequently, extends the spectrum of        tumor-specific targets and may be of substantial therapeutic        value. Second and third generation CARs have been designed to        provide therapeutic T cells with activation and costimulation        signals upon engaging a single antigen through the tethering of        two or more signaling portions at the CAR endodomain. However,        if activation and costimulation are split in the same T-cell        between two CARs, each specific for a different antigen, then        full blown response would require the cooperation of the two        complementary signals that could only be accomplished in the        presence of the two antigens. This principle has been        demonstrated in several preclinical studies (Kloss et al., 2013;        Lanitis et al., 2013; Wilkie et al., 2012; WO 2016/126608).

While undoubtedly intriguing, this approach still faces the need inmeticulous titration of the magnitude of both the activating andcostimulatory signals so as to reach the optimal balance that would onlyallow effective on-target, on-tumor T cell reactivity. Whether suchbalance can be routinely attained in the clinical setting is stillquestionable.

An entirely new approach for limiting T cell response only to targetcells that express a unique combination of two antigens was publishedrecently (Roybal et al., 2016a). Its core element functions as a‘genetic switch’ which exploits the mode of action of several cellsurface receptors, including Notch. Following binding of such a receptorto its ligand it undergoes dual cleavage resulting in the liberation ofits intracellular domain which translocates to the cell nucleus where itfunctions as a transcription factor. The implementation of thisprinciple entails the co-introduction of two genes to the effector Tcells. The first one is expressed constitutively and encodes such achimeric cleavable receptor equipped with a recognition moiety directedat the first antigen. Engagement with this antigen on the surface of atarget cell will turn on the expression of the second gene encoding aconventional CAR which is directed at the second antigen. The targetcell will be killed only if it co-expresses this second antigen as well.

Inhibitory CARs. Off-tumor reactivity occurs when the target antigen ofCAR-redirected killer cells is shared with normal tissue. If this normaltissue expresses another surface antigen not present on the tumor, thenco-expressing in the gene-modified cells an additional CAR targetingthis non-shared antigen, which harbors an inhibitory signaling moiety,can prevent T-cell activation by the normal tissue.

Instead of an activating domain (such as FcRy or CD3-ζ), an iCARpossesses a signaling domain derived from an inhibitory receptor whichcan antagonize T cell activation, such as CTLA-4, PD-1 or an NKinhibitory receptor. If the normal tissue which shares the candidateaCAR antigen with the tumor expresses another surface antigen not sharedwith the tumor, an iCAR expressed by the same T cell which targets thisnon-shared antigen can protect the normal tissue (FIG. 1).

Unlike T cells, each of which expresses a unique two-chain TCR encodedby somatically rearranged gene segments, NK cells do not expressantigen-specific receptors. Instead, NK cells express an array ofgermline-encoded activating and inhibitory receptors which respectivelyrecognize multiple activating and inhibitory ligands at the cell surfaceof infected and healthy cells. The protective capacity of an iCAR basedon NK inhibitory receptors such as KIR3DL1 has been described (U.S. Pat.No. 9,745,368). KIR3DL1 and other NK inhibitory receptors function bydismantling the immunological synapse in a rapid and comprehensivemanner. There is compelling evidence that a single NK cell can spare aresistant cell expressing both inhibitory and activating ligands yetkill a susceptible cell it simultaneously engages, which expresses onlythe activating ligands (Abeyweera et al., 2011; Eriksson et al., 1999;Treanor et al., 2006; Vyas et al., 2001). This exquisite ability isgoverned by the different spatial organization of signal transductionmolecules formed at each of the respective immune synapses whichconsequently affects the exocytosis of cytolytic granules (see (Huse etal., 2013) for review). More recently, Fedorov et al. (Fedorov et al.,2013a; WO 2015/142314) successfully employed for this purpose theintracellular domains of PD-1 and CTLA-4. Unlike NK inhibitoryreceptors, the regulatory effects of these iCARs affected the entirecell. Yet, these effects were temporary, allowing full T-cell activationupon subsequent encounter with target cells expressing only the aCARantigen.

Tissue distribution of the antigens targeted by the iCAR and aCARdictates the optimal mode of action of the iCAR required for conferringmaximal safety without compromising clinical efficacy. For example, ifthe anatomical sites of the tumor and the normal tissue(s) to beprotected do not intersect, transient inhibition (CTLA-4- or PD-1-like)will likely suffice. Yet, if these sites do overlap, onlysynapse-confined inhibition (e.g., an NK mode of action) will preventconstant paralysis of the therapeutic cells and allow their effectivetumoricidal activity. The approach of using iCARs to reduce on-targetoff-tumor reactivity suffers from a dire lack of antigens downregulatedin tumor cells but present on normal tissue.

Next generation sequencing (NGS) allows the determination of the DNAsequence of all protein-coding genes (˜1% of the entire genome) in agiven tumor biopsy and the comparison of the cancer ‘exome’ to that of ahealthy tissue (usually from white blood cells) of the same patient.Exome sequencing can be completed within several days post-biopsyremoval and at relatively low cost. In parallel, transcriptome analysis(RNA-seq) can provide complementary information on the genes that areactually expressed by the same cell sample.

It is becoming increasingly clear that the mutational landscape of eachindividual tumor is unique (Lawrence et al., 2013; Vogelstein et al.,2013). As a result of nonsynonymous mutations the tumor cell canpotentially present a private set of neopeptides to the patient's immunesystem on one or more of his or her HLA products. Indeed, tremendousefforts are being put in recent years into identifying tumor-specificneoepitopes which can be recognized by the patient's own CD8 or CD4 Tcell repertoire and serve as targets for immunotherapy (for review see(Blankenstein et al., 2015; Van Buuren et al., 2014; Heemskerk et al.,2013; Overwijk et al., 2013; Schumacher and Schreiber, 2015)). However,cumulative findings suggest that neoantigen-based T cell immunotherapiesare more likely to be effective in cancers displaying higher mutationalload, such as melanoma and lung cancers, but may often fail to showbenefit in most cancers with fewer mutations (Savage, 2014; Schumacherand Schreiber, 2015). Furthermore, considerable intratumoralheterogeneity (Burrell et al., 2013) entails the simultaneousco-targeting of several antigens so as to avoid emergence ofmutation-loss variants, a task which becomes increasingly demanding inview of the scarcity of useful immunogenic neopeptides.

All in all, the urgent need to identify suitable targets for cancerimmunotherapy via the adoptive transfer of genetically redirected killercells is still largely unmet.

BRIEF SUMMARY OF THE INVENTION

In some aspects, the present invention provides a method of identifyinga target for preparing an inhibitory chimeric antigen receptor (iCAR) ora protective chimeric antigen receptor (pCAR) capable of preventing orattenuating undesired activation of an effector immune cell, wherein thetarget is identified by a method comprising:

-   -   (i) identifying a gene with at least two expressed alleles that        encodes a protein comprising an extracellular polymorphic        epitope;    -   (ii) determining that at least one of the expressed alleles        exhibits an amino acid sequence change in the extracellular        polymorphic epitope sequence relative to an extracellular        polymorphic epitope reference sequence;    -   (iii) determining that the gene is located in a chromosomal        region which undergoes loss of heterozygosity (LOH) in a tumor        type; and    -   (iv) determining that the gene is expressed in the        tissue-of-origin of the tumor type in which the chromosomal        region was found to undergo LOH.

In some embodiments, the LOH position is selected from the groupconsisting of a substitution, deletion, and insertion. In someembodiments, the LOH position is a SNP. In some embodiments, the genecomprising the extracellular polymorphic epitope is an HLA gene.

In some embodiments, the gene comprising the extracellular polymorphicepitope is an HLA-A, HLA-B, HLA-C, HLA-G, HLA-E, HLA-F, HLA-K, HLA-L,HLA-DM, HLA-DO, HLA-DP, HLA_DQ, or HLA-DR gene. In some embodiments, thegene comprising the extracellular polymorphic epitope is an HLA-A gene.In some embodiments, the gene comprising the extracellular polymorphicepitope is an HLA-B gene. In some embodiments, the gene comprising theextracellular polymorphic epitope is an HLA-C gene. In some embodiments,the gene comprising the extracellular polymorphic epitope is an HLA-Ggene. In some embodiments, the gene comprising the extracellularpolymorphic epitope is an HLA-E gene. In some embodiments, the genecomprising the extracellular polymorphic epitope is an HLA-F gene. Insome embodiments, the gene comprising the extracellular polymorphicepitope is an HLA-K gene. In some embodiments, the gene comprising theextracellular polymorphic epitope is an HLA-L gene. In some embodiments,the gene comprising the extracellular polymorphic epitope is an HLA-DMgene. In some embodiments, the gene comprising the extracellularpolymorphic epitope is an HLA-DO gene. In some embodiments, theextracellular polymorphic epitope is an HLA-DP gene. In someembodiments, the extracellular polymorphic epitope is an HLA_DQ gene. Insome embodiments, the extracellular polymorphic epitope is an HLA-DRgene.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 1. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ABCA4, ADAM30, AQP10, ASTN1, C1orf101, CACNA1S,CATSPER4, CD101, CD164L2, CD1A, CD1C, CD244, CD34, CD46, CELSR2, CHRNB2,CLCA2, CLDN19, CLSTN1, CR1, CR2, CRB1, CSF3R, CSMD2, ECE1, ELTD1, EMC1,EPHA10, EPHA2, EPHA8, ERMAP, FCAMR, FCER1A, FCGR1B, FCGR2A, FCGR2B,FCGR3A, FCRL1, FCRL3, FCRL4, FCRL5, FCRL6, GJB4, GPA33, GPR157, GPR37L1,GPR88, HCRTR1, IGSF3, IGSF9, IL22RA1, IL23R, ITGA10, KIAA1324, KIAA2013,LDLRAD2, LEPR, LGR6, LRIG2, LRP8, LRRC52, LRRC8B, LRRN2, LY9, MIA3, MR1,MUC1, MXRA8, NCSTN, NFASC, NOTCH2, NPR1, NTRK1, OPN3, OR10J1, OR10J4,OR10K1, OR1OR2, OR10T2, OR10X1, OR11L1, OR14A16, OR14I1, OR14K1, OR2AK2,OR2C3, OR2G2, OR2G3, OR2L2, OR2M7, OR2T12, OR2T27, OR2T1, OR2T3, OR2T29,OR2T33, OR2T34, OR2T35, OR2T3, OR2T4, OR2T5, OR2T6, OR2T7, OR2T8, OR2W3,OR6F1, OR6K2, OR6K3, OR6K6, OR6N1, OR6P1, OR6Y1, PDPN, PEAR1, PIGR,PLXNA2, PTCH2, PTCHD2, PTGFRN, PTPRC, PTPRF, PTGFRN, PVRL4, RHBG, RXFP4,S1PR1, SCNN1D, SDC3, SELE, SELL, SELP, SEMA4A, SEMA6C, SLAMF7, SLAMF9,SLC2A7, SLC5A9, TACSTD2, TAS1R2, TIE1, TLR5, TMEM81, TNFRSF14, TNFRSF1B,TRABD2B, USH2A, VCAM1, and ZP4.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 2. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ABCG5, ALK, ASPRV1, ATRAID, CD207, CD8B, CHRNG,CLEC4F, CNTNAP5, CRIM1, CXCR1, DNER, DPP10, EDAR, EPCAM, GPR113, GPR148,GPR35, GPR39, GYPC, IL1RL1, ITGA4, ITGA6, ITGAV, LCT, LHCGR, LRP1B,LRP2, LY75, MARCO, MERTK, NRP2, OR6B2, PLA2R1, PLB1, PROKR1, PROM2,SCN7A, SDC1, SLC23A3, SLC5A6, TGOLN2, THSD7B, TM4SF20, TMEFF2, TMEM178A,TPO, and TRABD2A.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 3. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ACKR2, ALCAM, ANO10, ATP13A4, BTLA, CACNA1D,CACNA2D2, CACNA2D3, CASR, CCRL2, CD200, CD200R1, CD86, CD96, CDCP1,CDHR4, CELSR3, CHL1, CLDN11, CLDN18, CLSTN2, CSPG5, CX3CR1, CXCR6,CYP8B1, DCBLD2, DRD3, EPHA6, EPHB3, GABRR3, GP5, GPR128, GPR15, GPR27,GRM2, GRM7, HEG1, HTR3C, HTR3D, HTR3E, IGSF11, IL17RC, IL17RD, IL17RE,IL5RA, IMPG2, ITGA9, ITGB5, KCNMB3, LRIG1, LRRC15, LRRN1, MST1R,NAALADL2, NRROS, OR5AC1, OR5H1, OR5H14, OR5H15, OR5H6, OR5K2, OR5K3,OR5K4, PIGX, PLXNB1, PLXND1, PRRT3, PTPRG, ROBO2, RYK, SEMA5B, SIDT1,SLC22A14, SLC33A1, SLC4A7, SLITRK3, STAB1, SUSD5, TFRC, TLR9, TMEM108,TMEM44, TMPRSS7, TNFSF10, UPK1B, VIPR1, and ZPLD1.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 4. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ANTXR2, BTC, CNGA1, CORIN, EGF, EMCN, ENPEP, EPHA5,ERVMER34-1, EVC2, FAT1, FAT4, FGFRL1, FRAS1, GPR125, GRID2, GYPA, GYPB,KDR, KIAA0922, KLB, MFSD8, PARM1, PDGFRA, RNF150, TENM3, TLR10, TLR1,TLR6, TMEM156, TMPRSS11A, TMPRSS11B, TMPRSS11E, TMPRSS11F, UGT2A1, andUNC5C.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 5. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ADAM19, ADRB2, BTNL3, BTNL8, BTNL9, C5orf15,CATSPER3, CD180, CDH12, CDHR2, COL23A1, CSF1R, F2RL2, FAM174A, FAT2,FGFR4, FLT4, GABRA6, GABRG2, GPR151, GPR98, GRM6, HAVCR1, HAVCR2,IL31RA, IL6ST, IL7R, IQGAP2, ITGA1, ITGA2, KCNMB1, LIFR, LNPEP, MEGF10,NIPAL4, NPR3, NRG2, OR2V1, OR2Y1, OSMR, PCDH12, PCDH1, PCDHA1, PCDHA2,PCDHA4, PCDHA8, PCDHA9, PCDHB10, PCDHB11, PCDHB13, PCDHB14, PCDHB15,PCDHB16, PCDHB2, PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHGA1, PCDHGA4,PDGFRB, PRLR, SEMA5A, SEMA6A, SGCD, SLC1A3, SLC22A4, SLC22A5, SLC23A1,SLC36A3, SLC45A2, SLC6A18, SLC6A19, SLCO6A1, SV2C, TENM2, TIMD4, andUGT3A1.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 6. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of BAI3, BTN1A1, BTN2A1, BTN2A2, BTN3A1, BTN3A2, BTNL2,CD83, DCBLD1, DLL1, DPCR1, ENPP1, ENPP3, ENPP4, EPHA7, GABBR1, GABRR1,GCNT6, GFRAL, GJB7, GLP1R, GPR110, GPR111, GPR116, GPR126, GPR63,GPRC6A, HFE, HLA-A, HLA-B, HLA-C, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1,HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-G,IL20RA, ITPR3, KIAA0319, LMBRD1, LRFN2, LRP11, MAS1L, MEP1A, MICA, MICB,MOG, MUC21, MUC22, NCR2, NOTCH4, OPRM1, OR10C1, OR12D2, OR12D3, OR14J1,OR2B2, OR2B6, OR2J1, OR2W1, OR5V1, PDE10A, PI16, PKHD1, PTCRA, PTK7,RAET1E, RAET1G, ROS1, SDIM1, SLC16A10, SLC22A1, SLC44A4, TAAR2, TREM1,TREML1, and TREML2.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 7. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of AQP1, C7orf50, CD36, CDHR3, CNTNAP2, DPP6, EGFR,EPHA1, EPHB6, ERVW-1, GHRHR, GJC3, GPNMB, GRM8, HUS1, HYAL4, KIAA1324L,LRRN3, MET, MUC12, MUC17, NPC1L1, NPSR1, OR2A12, OR2A14, OR2A25, OR2A42,OR2A7, OR2A2, OR2AE1, OR2F2, OR6V1, PILRA, PILRB, PKD1L1, PLXNA4, PODXL,PTPRN2, PTPRZ1, RAMP3, SLC29A4, SMO, TAS2R16, TAS2R40, TAS2R4, TFR2,THSD7A, TMEM213, TTYH3, ZAN, and ZP3.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 8. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ADAM18, ADAM28, ADAM32, ADAM7, ADAM9, ADRA1A, CDH17,CHRNA2, CSMD1, CSMD3, DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1,PRSS55, SCARA3, SCARA5, SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A1,TNFRSF10A, and TNFRSF10B.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 9. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ABCA1, AQP7, ASTN2, C9orf135, CA9, CD72, CNTNAP3B,CNTNAP3, CRB2, ENTPD8, GPR144, GRIN3A, IZUMO3, KIAA1161, MAMDC4, MEGF9,MUSK, NOTCH1, OR13C2, OR13C3, OR13C5, OR13C8, OR13C9, OR13D1, OR13F1,OR1B1, OR1J2, OR1K1, OR1L1, OR1L3, OR1L6, OR1L8, OR1N1, OR1N2, OR1Q1,OR2S2, PCSK5, PDCD1LG2, PLGRKT, PTPRD, ROR2, SEMA4D, SLC31A1, TEK, TLR4,TMEM2, and VLDLR.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 10. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ABCC2, ADAM8, ADRB1, ANTXRL, ATRNL1, C10orf54,CDH23, CDHR1, CNNM2, COL13A1, COL17A1, ENTPD1, FZD8, FGFR2, GPR158,GRID1, IL15RA, IL2RA, ITGA8, ITGB1, MRC1, NRG3, NPFFR1, NRP1, OPN4,PCDH15, PKD2L1, PLXDC2, PRLHR, RET, RGR, SLC16A9, SLC29A3, SLC39A12,TACR2, TCTN3, TSPAN15, UNC5B, and VSTM4.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 11. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of AMICA1, ANO1, ANO3, APLP2, C11orf24, CCKBR, CD248,CD44, CD5, CD6, CD82, CDON, CLMP, CRTAM, DCHS1, DSCAML1, FAT3, FOLH1,GDPD4, GDPD5, GRIK4, HEPHL1, HTR3B, IFITM10, IL10RA, KIRREL3, LGR4,LRP4, LRP5, LRRC32, MCAM, MFRP, MMP26, MPEG1, MRGPRE, MRGPRF, MRGPRX2,MRGPRX3, MRGPRX4, MS4A4A, MS4A6A, MTNR1B, MUC15, NAALAD2, NAALADL1,NCAM1, NRXN2, OR10A2, OR10A5, OR10A6, OR10D3, OR10G4, OR10G7, OR10G8,OR10G9, OR10Q1, OR10S1, OR1S1, OR2AG1, OR2AG2, OR2D2, OR4A47, OR4A15,OR4A5, OR4C11, OR4C13, OR4C15, OR4C16, OR4C3, OR4C46, OR4C5, OR4D6,OR4A8P, OR4D9, OR4S2, OR4X1, OR51E1, OR51L1, OR52A1, OR52E1, OR52E2,OR52E4, OR52E6, OR52I1, OR52I2, OR52J3, OR52L1, OR52N1, OR52N2, OR52N4,OR52W1, OR56B1, OR56B4, OR5A1, OR5A2, OR5AK2, OR5AR1, OR5B17, OR5B3,OR5D14, OR5D16, OR5D18, OR5F1, OR5I1, OR5L2, OR5M11, OR5M3, OR5P2,OR5R1, OR5T2, OR5T3, OR5W2, OR6A2, OR6T1, OR6X1, OR8A1, OR8B12, OR8B2,OR8B3, OR8B4, OR8D1, OR8D2, OR8H1, OR8H2, OR8H3, OR8I2, OR8J1, OR8J2,OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR9G1, OR9G4, OR9Q2, P2RX3, PTPRJ,ROBO3, SIGIRR, SLC22A10, SLC3A2, SLC5A12, SLCO2B1, SORL1, ST14, SYT8,TENM4, TMEM123, TMEM225, TMPRSS4, TMPRSS5, TRIM5, TRPM5, TSPAN18, andZP1.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 12. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ANO4, AVPR1A, BCL2L14, CACNA2D4, CD163, CD163L1,CD27, CD4, CLEC12A, CLEC1B, CLEC2A, CLEC4C, CLEC7A, CLECL1, CLSTN3,GPR133, GPRC5D, ITGA7, ITGB7, KLRB1, KLRC2, KLRC3, KLRC4, KLRF1, KLRF2,LRP1, LRP6, MANSC1, MANSC4, OLR1, OR10AD1, OR10P1, OR2AP1, OR6C1, OR6C2,OR6C3, OR6C4, OR6C6, OR6C74, OR6C76, OR8S1, OR9K2, ORAI1, P2RX4, P2RX7,PRR4, PTPRB, PTPRQ, PTPRR, SCNN1A, SELPLG, SLC2A14, SLC38A4, SLC5A8,SLC6A15, SLC8B1, SLCO1A2, SLCO1B1, SLCO1B7, SLCO1C1, SSPN, STAB2,TAS2R10, TAS2R13, TAS2R14, TAS2R20, TAS2R30, TAS2R31, TAS2R42, TAS2R43,TAS2R46, TAS2R7, TMEM119, TMEM132B, TMEM132C, TMEM132D, TMPRSS12,TNFRSF1A, TSPAN8, and VSIG10.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 13. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ATP4B, ATP7B, FLT3, FREM2, HTR2A, KL, PCDH8, RXFP2,SGCG, SHISA2, SLC15A1, SLITRK6, and TNFRSF19.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 14. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ADAM21, BDKRB2, C14orf37, CLEC14A, DLK1, FLRT2,GPR135, GPR137C, JAG2, LTB4R2, MMP14, OR11G2, OR11H12, OR11H6, OR4K1,OR4K15, OR4K5, OR4L1, OR4N2, OR4N5, SLC24A4, and SYNDIG1L.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 15. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ANPEP, CD276, CHRNA7, CHRNB4, CSPG4, DUOX1, DUOX2,FAM174B, GLDN, IGDCC4, ITGA11, LCTL, LTK, LYSMD4, MEGF11, NOX5, NRG4,OCA2, OR4F4, OR4M2, OR4N4, PRTG, RHCG, SCAMPS, SEMA4B, SEMA6D, SLC24A1,SLC24A5, SLC28A1, SPG11, STRA6, TRPM1, and TYRO3.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 16. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ATP2C2, CACNA1H, CD19, CDH11, CDH15, CDH16, CDH3,CDH5, CNGB1, CNTNAP4, GDPD3, GPR56, GPR97, IFT140, IL4R, ITFG3, ITGAL,ITGAM, ITGAX, KCNG4, MMP15, MSLNL, NOMO1, NOMO3, OR2C1, PIEZO1, PKD1,PKD1L2, QPRT, SCNN1B, SEZ6L2, SLC22A31, SLC5A11, SLC7A6, SPN, TMC5,TMC7, TMEM204, TMEM219, and TMEM8A.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 17. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ABCC3, ACE, AOC3, ARL17B, ASGR2, C17orf80, CD300A,CD300C, CD300E, CD300LF, CD300LG, CHRNB1, CLEC10A, CNTNAP1, CPD, CXCL16,ERBB2, FAM171A2, GCGR, GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3,ITGAE, ITGB3, KCNJ12, LRRC37A2, LRRC37A3, LRRC37A, LRRC37B, MRC2, NGFR,OR1A2, OR1D2, OR1G1, OR3A1, OR3A2, OR4D1, OR4D2, RNF43, SCARF1, SCN4A,SDK2, SECTM1, SEZ6, SHPK, SLC26A11, SLC5A10, SPACA3, TMEM102, TMEM132E,TNFSF12, TRPV3, TTYH2, and TUSC5.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 18. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of APCDD1, CDH19, CDH20, CDH7, COLEC12, DCC, DSC1,DSG1, DSG3, DYNAP, MEP1B, PTPRM, SIGLEC15, and TNFRSF11A.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 19. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ABCA7, ACPT, BCAM, C19orf38, C19orf59, C5AR1,CATSPERD, CATSPERG, CD22, CD320, CD33, CD97, CEACAM19, CEACAM1,CEACAM21, CEACAM3, CEACAM4, CLEC4M, DLL3, EMR1, EMR2, EMR3, ERVV-1,ERVV-2, FAM187B, FCAR, FFAR3, FPR1, FXYD5, GFY, GP6, GPR42, GRIN3B,ICAM3, IGFLR1, IL12RB1, IL27RA, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1,KIR3DL2, KIR3DL3, KIRREL2, KISS1R, LAIR1, LDLR, LILRA1, LILRA2, LILRA4,LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LINGO3, LPHN1, LRP3,MADCAM1, MAG, MEGF8, MUC16, NCR1, NOTCH3, NPHS1, OR10H1, OR10H2, OR10H3,OR10H4, OR1I1, OR2Z1, OR7A10, OR7C1, OR7D4, OR7E24, OR7G1, OR7G2, OR7G3,PLVAP, PTGIR, PTPRH, PTPRS, PVR, SCN1B, SHISA7, SIGLEC10, SIGLEC11,SIGLEC12, SIGLEC5, SIGLEC6, SIGLEC8, SIGLEC9, SLC44A2, SLC5A5, SLC7A9,SPINT2, TARM1, TGFBR3L, TMC4, TMEM91, TMEM161A, TMPRSS9, TNFSF14,TNFSF9, TRPM4, VN1R2, VSIG10L, VSTM2B, and ZNRF4.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 20. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ABHD12, ADAM33, ADRA1D, APMAP, ATRN, CD40, CD93,CDH22, CDH26, CDH4, FLRT3, GCNT7, GGT7, JAG1, LRRN4, NPBWR2, OCSTAMP,PTPRA, PTPRT, SEL1L2, SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3, SLC2A10,SLC4A11, SSTR4, and THBD.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 21. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of CLDN8, DSCAM, ICOSLG, IFNAR1, IFNGR2, IGSF5, ITGB2,KCNJ15, NCAM2, SLC19A1, TMPRSS15, TMPRSS2, TMPRSS3, TRPM2, and UMODL1.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 22. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of CACNA1I, CELSR1, COMT, CSF2RB, GGT1, GGT5, IL2RB,KREMEN1, MCHR1, OR11H1, P2RX6, PKDREJ, PLXNB2, SCARF2, SEZ6L, SSTR3,SUSD2, TMPRSS6, and TNFRSF13C.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome X. In some embodiments, the genecomprising the extracellular polymorphic epitope is selected from thegroup consisting of ATP6AP2, ATP7A, CNGA2, EDA2R, FMR1NB, GLRA4, GPR112,GUCY2F, HEPH, P2RY10, P2RY4, PLXNA3, PLXNB3, TLR8, VSIG4, and XG.

In some embodiments, the tumor is selected from the group consisting ofa breast tumor, a prostate tumor, an ovarian tumor, a cervical tumor, askin tumor, a pancreatic tumor, a colorectal tumor, a renal tumor, aliver tumor, a brain tumor, a lymphoma, a leukemia, a lung tumor, and aglioma.

In some embodiments, the tumor is selected from the group consisting ofan adrenal gland tumor, a kidney tumor, a melanoma, DLBC, a breasttumor, a sarcoma, an ovary tumor, a lung tumor, a bladder tumor, and aliver tumor. In some embodiments, the adrenal gland tumor is anadrenocortical carcinoma. In some embodiments, the kidney tumor is achromophobe renal cell carcinoma. In some embodiments, the melanoma isuveal melanoma.

The present invention also provides safe effector cells. In someembodiments, the present invention provides a safe effector immune cellexpressing (i) an iCAR or pCAR according to any of claims 1 through 46and (ii) an activating chimeric antigen receptor (aCAR).

In some embodiments, the safe effector immune cell of claim 47, whereinthe aCAR is directed against or specifically binds to a tumor-associatedantigen or a non-polymorphic cell surface epitope. In some embodiments,due to the protective effects of the iCAR or pCAR, the aCAR can bedirected against any surface protein expressed on a cancer cell.

In some embodiments, the the aCAR is directed against or specificallybinds to a tumor associated protein, a CAR target as listed in table 1,any cell surface protein that is expressed in a tumor tissue in whichthe iCAR is also expressed.

In some embodiments, the non-polymorphic cell surface epitope isselected from the group consisting of CD19, CD20, CD22, CD10, CD7,CD49f, CD56, CD74, CAIX Igκ, ROR1, ROR2, CD30, LewisY, CD33, CD34, CD38,CD123, CD28, CD44v6, CD44, CD41, CD133, CD138, NKG2D-L, CD139, BCMA,GD2, GD3, hTERT, FBP, EGP-2, EGP-40, FR-α, L1-CAM, ErbB2,3,4, EGFRvIII,VEGFR-2, IL-13Rα2, FAP, Mesothelin, c-MET, PSMA, CEA, kRas, MAGE-A1,MUC1, MUC16, PDL1, PSCA, EpCAM, FSHR, AFP, AXL, CD80, CD89, CDH17,CLD18, GPC3, TEM8, TGFB1, NY-ESO-1, WT-1 and EGFR.

51. The safe effector immune cell of any of claims 47 to 50, wherein thesafe effector immune cell is an autologous or a universal (allogeneic)effector cell.

In some embodiments, the safe effector immune cell is selected from thegroup consisting of a T cell, a natural killer cell and acytokine-induced killer cell.

In some embodiments of the safe effector immune cell, the expressionlevel of the iCAR or pCAR is greater than or equal to the expressionlevel of the aCAR.

In some embodiments of the safe effector immune cell, the iCAR or pCARis expressed by a first vector and the aCAR is expressed by a secondvector.

In some embodiments of the safe effector immune cell, the iCAR or pCARand the aCAR are both expressed by the same vector.

In some embodiments of the safe effector immune cell, the nucleotidesequence encoding for the aCAR is downstream of the nucleotide sequenceencoding for the iCAR or pCAR.

In some embodiments of the safe effector immune cell, the nucleotidesequence comprises a viral self-cleaving 2A peptide between thenucleotide sequence encoding for the aCAR and the nucleotide sequenceencoding for the iCAR or pCAR.

In some embodiments of the safe effector immune cell, the viralself-cleaving 2A peptide is selected from the group consisting of T2Afrom Thosea asigna virus (TaV), F2A from Foot-and-mouth disease virus(FMDV), E2A from Equine rhinitis A virus (ERAV) and P2A from Porcineteschovirus-1 (PTV1).

In some embodiments of the safe effector immune cell, the nucleotidesequence encoding the aCAR is linked via a flexible linker to the iCARor pCAR.

In some embodiments of the safe effector immune cell, the aCAR comprisesat least one signal transduction element that activates or co-stimulatesan effector immune cell.

In some embodiments of the safe effector immune cell, the at least onesignal transduction element that activates or co-stimulates an effectorimmune cell is homolgous to an immunoreceptor tyrosine-based activationmotif (ITAM) of for example CD3ζ or FcRγ chains.

In some embodiments of the safe effector immune cell, the at least onesignal transduction element that activates or co-stimulates an effectorimmune cell is homolgous to an activating killer cellimmunoglobulin-like receptor (KIR), such as KIR2DS and KIR3DS.

In some embodiments of the safe effector immune cell, the at least onesignal transduction element that activates or co-stimulates an effectorimmune cell is homolgous to or an adaptor molecule, such as DAP12.

In some embodiments of the safe effector immune cell, the at least onesignal transduction element that activates or co-stimulates an effectorimmune cell is homolgous to or a co-stimulatory signal transductionelement of CD27, CD28, ICOS, CD137 (4-1BB), CD134 (OX40) or GITR.

The present invention also provides a method for treating cancer in apatient having a tumor characterized by LOH, comprising administering tothe patient a safe effector immune cell expressing an iCAR as describedherein.

In some embodiments, the invention further provides a method fortreating cancer in a patient having a tumor characterized by LOH,comprising administering to the patient a safe effector immune cell asdescribed herein.

In one aspect, the present invention provides a nucleic acid moleculecomprising a nucleotide sequence encoding an inhibitory chimeric antigenreceptor (iCAR) capable of preventing or attenuating undesiredactivation of an effector immune cell, wherein the iCAR comprises anextracellular domain that specifically binds to a single allelic variantof a polymorphic cell surface epitope absent from mammalian tumor cellsdue to loss of heterozygosity (LOH) but present at least on all cells ofrelated mammalian normal tissue and on vital organs; and anintracellular domain comprising at least one signal transduction elementthat inhibits an effector immune cell. In some embodiments, the iCAR orpCAR target is expressed on all cells that the aCAR target is normallyexpressed in. In some embodiments, the iCAR or pCAR target is expressedin the vital organ cells the aCAR is expressed in.

In an additional aspect, the present invention provides a vectorcomprising a nucleic acid molecule of the invention as defined herein,and at least one control element, such as a promoter, operably linked tothe nucleic acid molecule.

In another aspect, the present invention provides a method of preparingan inhibitory chimeric antigen receptor (iCAR) capable of preventing orattenuating undesired activation of an effector immune cell, accordingto the present invention as defined herein, the method comprising: (i)retrieving a list of human genomic variants of protein-encoding genesfrom at least one database of known variants; (ii) filtering the list ofvariants retrieved in (i) by: (a) selecting variants resulting in anamino acid sequence variation in the protein encoded by the respectivegene as compared with its corresponding reference allele, (b) selectingvariants of genes wherein the amino acid sequence variation is in anextracellular domain of the encoded protein, (c) selecting variants ofgenes that undergo loss of heterozygosity (LOH) at least in one tumor,and (d) selecting variants of genes that are expressed at least in atissue of origin of the at least one tumor in which they undergo LOHaccording to (c), thereby obtaining a list of variants having an aminoacid sequence variation in an extracellular domain in the proteinencoded by the respective gene lost in the at least one tumor due to LOHand expressed at least in a tissue of origin of the at least one tumor;(iii) defining a sequence region comprising at least one single variantfrom the list obtained in (ii), sub-cloning and expressing the sequenceregion comprising the at least one single variant and a sequence regioncomprising the corresponding reference allele thereby obtaining therespective epitope peptides; (iv) selecting an iCAR binding domain,which specifically binds either to the epitope peptide encoded by thecloned sequence region, or to the epitope peptide encoded by thecorresponding reference allele, obtained in (iii); and (vii) preparingiCARs as defined herein, each comprising an iCAR binding domain asdefined in (iv).

In still another aspect, the present invention provides a method forpreparing a safe effector immune cell comprising: (i) transfecting aTCR-engineered effector immune cell directed to a tumor-associatedantigen with a nucleic acid molecule comprising a nucleotide sequenceencoding an iCAR as defined herein or transducing the cells with avector defined herein; or (ii) transfecting a naïve effector immune cellwith a nucleic acid molecule comprising a nucleotide sequence encodingan iCAR as defined herein and a nucleic acid molecule comprising anucleotide sequence encoding an aCAR as defined herein; or transducingan effector immune cell with a vector as defined herein.

In yet another aspect, the present invention provides a safe effectorimmune cell obtained by the method of the present invention as describedherein. The safe effector immune cell may be a redirected T cellexpressing an exogenous T cell receptor (TCR) and an iCAR, wherein theexogenous TCR is directed to a non-polymorphic cell surface epitope ofan antigen or a single allelic variant of a polymorphic cell surfaceepitope, wherein said epitope is a tumor-associated antigen or is sharedat least by cells of related tumor and normal tissue, and the iCAR is asdefined herein; or the safe effector immune cell is a redirectedeffector immune cell such as a natural killer cell or a T cellexpressing an iCAR and an aCAR as defined herein.

In a further aspect, the present invention provides a method ofselecting a personalized biomarker for a subject having a tumorcharacterized by LOH, the method comprising (i) obtaining a tumor biopsyfrom the subject; (ii) obtaining a sample of normal tissue from thesubject, e.g., peripheral blood mononuclear cells (PBMCs); and (iii)identifying a single allelic variant of a polymorphic cell surfaceepitope that is not expressed by cells of the tumor due to LOH, but thatis expressed by the cells of the normal tissue, thereby identifying apersonalized biomarker for the subject.

In a further aspect, the present invention provides a method fortreating cancer in a patient having a tumor characterized by LOH,comprising administering to the patient an effector immune cell asdefined herein, wherein the iCAR is directed to a single allelic variantencoding a polymorphic cell surface epitope absent from cells of thetumor due to loss of heterozygosity (LOH) but present at least on allcells of related mammalian normal tissue of the patient.

In still a further aspect, the present invention is directed to a safeeffector immune cell as defined herein for use in treating a patienthaving a tumor characterized by LOH, wherein the iCAR is directed to asingle allelic variant encoding a polymorphic cell surface epitopeabsent from cells of the tumor due to loss of heterozygosity (LOH) butpresent at least on all cells of related mammalian normal tissue of thepatient, including the vital organs of the patient. In some embodiments,the iCAR or pCAR is expressed on all cells that the aCAR target isnormally expressed in. In some embodiments, the iCAR or pCAR isexpressed in vital organ cells that the aCAR is expressed in.

In yet a further aspect, the present invention is directed to a methodfor treating cancer in a patient having a tumor characterized by LOHcomprising: (i) identifying or receiving information identifying asingle allelic variant of a polymorphic cell surface epitope that is notexpressed by cells of the tumor due to LOH, but that is expressed by thecells of the normal tissue, (ii) identifying or receiving informationidentifying a non-polymorphic cell surface epitope of an antigen or asingle allelic variant of a polymorphic cell surface epitope, whereinsaid epitope is a tumor-associated antigen or is shared by cells atleast of related tumor and normal tissue in said cancer patient; (iii)selecting or receiving at least one nucleic acid molecule defining aniCAR as defined herein and at least one nucleic acid molecule comprisinga nucleotide sequence encoding an aCAR as defined herein, or at leastone vector as defined herein, wherein the iCAR comprises anextracellular domain that specifically binds to a cell surface epitopeof (i) and the aCAR comprises an extracellular domain that specificallybinds to a cell surface epitope of (ii); (iv) preparing or receiving atleast one population of safe redirected effector immune cells bytransfecting effector immune cells with the nucleic acid molecules of(iii) or transducing effector immune cells with the vectors of (iii);and (v) administering to said cancer patient at least one population ofsafe redirected immune effector cells of (iv).

In a similar aspect, the present invention provides at least onepopulation of safe redirected immune effector cells for treating cancerin a patient having a tumor characterized by LOH, wherein the saferedirected immune cells are obtained by (i) identifying or receivinginformation identifying a single allelic variant of a polymorphic cellsurface epitope that is not expressed by cells of the tumor due to LOH,but that is expressed by the cells of the normal tissue, (ii)identifying or receiving information identifying a non-polymorphic cellsurface epitope of an antigen or a single allelic variant of apolymorphic cell surface epitope, wherein said epitope is atumor-associated antigen or is shared by cells at least of related tumorand normal tissue in said cancer patient; (iii) selecting or receivingat least one nucleic acid molecule defining an iCAR as defined hereinand at least one nucleic acid molecule comprising a nucleotide sequenceencoding an aCAR as defined herein, or at least one vector as definedherein, wherein the iCAR comprises an extracellular domain thatspecifically binds to a cell surface epitope of (i) and the aCARcomprises an extracellular domain that specifically binds to a cellsurface epitope of (ii); (iv) preparing or receiving at least onepopulation of safe redirected effector immune cells by transfectingeffector immune cells with the nucleic acid molecules of (iii) ortransducing effector immune cells with the vectors of (iii).

In another aspect, the present invention is directed to a combination oftwo or more nucleic acid molecules, each one comprising a nucleotidesequence encoding a different member of a controlled effector immunecell activating system, said nucleic acid molecules being part of orforming a single continues nucleic acid molecule, or comprising two ormore separate nucleic acid molecules, wherein the controlled effectorimmune activating system directs effector immune cells to kill tumorcells that have lost one or more chromosomes or fractions thereof due toLoss of Heterozygosity (LOH) and spares cells of related normal tissue,and wherein (a) the first member comprises an activating chimericantigen receptor (aCAR) polypeptide comprising a first extracellulardomain that specifically binds to a non-polymorphic cell surface epitopeof an antigen or to a single allelic variant of a different polymorphiccell surface epitope and said non-polymorphic or polymorphic cellsurface epitope is a tumor-associated antigen or is shared by cells ofrelated abnormal and normal mammalian tissue; and (b) the second membercomprises a regulatory polypeptide comprising a second extracellulardomain that specifically binds to a single allelic variant of apolymorphic cell surface epitope not expressed by an abnormal mammaliantissue due to LOH but present on all cells of related mammalian normaltissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the concept of iCARs (taken from (Fedorov et al., 2013a).

FIG. 2 shows the aCAR/pCAR molecular design and mode of action. Bindingof the pCAR to its antigen on normal cells, whether these express theaCAR antigen or not, is expected to result in rapid RIP and breaking ofthe polypeptide into 3 separate fragments.

FIGS. 3A-C show the percentage of tumor samples undergoing LOH in thechromosomal region coding for the HLA class I locus. A. HLA-G, B. HLA-A,C. ZNRD1, in tumor types from the TCGA database. Kidney Chromophobe[KICH], Adrenocortical carcinoma [ACC], Pancreatic adenocarcinoma[PAAD], Sarcoma [SARC], Kidney renal papillary cell carcinoma [KIRP],Esophageal carcinoma [ESCA], Lung squamous cell carcinoma [LUSC], Kidneyrenal clear cell carcinoma [KIRC], Bladder Urothelial Carcinoma [BLCA],Ovarian serous cystadenocarcinoma [OV], Thymoma [THYM], Cervicalsquamous cell carcinoma and endocervical adenocarcinoma [CESC], Head andNeck squamous cell carcinoma [HNSC], Breast invasive carcinoma [BRCA],Stomach adenocarcinoma [STAD], Lymphoid Neoplasm Diffuse Large B-cellLymphoma [DLBC], Glioblastoma multiforme [GBM], Colon adenocarcinoma[COAD], Rectum adenocarcinoma [READ], Lung adenocarcinoma [LUAD],Testicular Germ Cell Tumors [TGCT], Mesothelioma [MESO],Cholangiocarcinoma [CHOL], Uterine Carcinosarcoma [UCS], Skin CutaneousMelanoma [SKCM], Uterine Corpus Endometrial Carcinoma [UCEC], BrainLower Grade Glioma [LGG], Prostate adenocarcinoma [PRAD], Liverhepatocellular carcinoma [LIHC], Thyroid carcinoma [THCA],Pheochromocytoma and Paraganglioma [PCPG], Acute Myeloid Leukemia[LAML], Uveal Melanoma [UVM]

FIG. 4 shows expression of HLA-A relative to all other protein codinggenes in the genome. The value for each gene reflects the mean RPKMvalue of tissue medians obtained from GTEX (gtexportal.org)

FIG. 5 shows a proposed workflow for analysis of HLA proteinloss-of-heterozygosity across cancers in Example 5.

FIG. 6 shows Frequency of LOH in the pancan12 dataset using ABSOLUTEprocessed copy number data. Lines represent 95% binomial confidenceintervals for frequency.

FIG. 7 shows the types of LOH observed in HLA-A. Of 588 episodes ofHLA-A LOH, none involved a breakpoint within the HLA-A gene.

FIG. 8 shows the distribution of length (in basepairs) of deletionsencompassing HLA-A. A large fraction of these deletions are greater thanthe length of chromosome 6p.

FIG. 9 shows the correlation between fraction of patients that have LOHof HLA-A in relative and ABSOLUTE copy number data with a threshold of−0.1.

FIG. 10A-10C shows the comparison of rate of LOH of HLA-A, HLA-B andHLA-C across 32 cancers reveals a nearly identical pattern of LOH.

FIG. 11 shows the IGV screenshot of AML copy number profiles sorted fordeletion of chromosome 6p. Blue indicates deletion, red indicatesamplification. There are no deletions of HLA-A.

FIG. 12 shows the proportion of uveal melanoma tumors undergoing LOH forall SNPs.

FIG. 13 provides the TCGA Study Abbreviations (also available athttps://gdc.cancer.gov/resources-tcga-users/tcga-code-tables/tcga-study-abbreviations).

FIG. 14 depicts the loss of a chromosomal region adjacent to the tumorsuppressor protein TP53, coded on chromosome 17. Genes coded onchromosome 17 which were identified as iCAR targets can be used to treatpatient RC001.

FIG. 15 provides a schematic diagram of iCAR and aCAR constructs.

FIG. 16 provides data regarding IL-2 secretion as measured by ELISA.iCAR specifically inhibits IL-2 secretion upon interaction with targetcells expressing iCAR target.

FIG. 17 shows that iCAR specifically inhibits IL-2 secretion uponinteraction with target cells expressing iCAR target as measured by CBA.

FIG. 18 shows specific activation of CD19 aCAR Jurkat-NFAT by CD19expressing target cells.

FIG. 19 shows specific inhibition of NFAT activation in CD19 aCAR/HLA-A2iCAR Jurkat-NFAT

FIG. 20 shows specific inhibition of NFAT activation at different E/Tratios.

FIG. 21 provides the sequences for the iCAR and aCAR constructs of FIG.15.

FIG. 22 provides the 1167 potential iCAR, pCAR and/or aCAR targets.

FIG. 23 provides the 3288 SNPs from the 1167 genes listed in FIG. 22.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

Referring to the revolutionary concept of tumor suppressor genes (TSGs)that had been put forward in 1971 by A. G. Knudson (Knudson Jr., 1971),Devilee, Cleton-Jansen and Cornelisse stated in the opening paragraph oftheir essay titled ‘Ever since Knudson’ (Devilee et al., 2001): “Manypublications have documented LOH on many different chromosomes in a widevariety of tumors, implicating the existence of multiple TSGs. Knudson'stwo-hit hypothesis predicts that these LOH events are the second step inthe inactivation of both alleles of a TSG”. In their seminal review ongenetic instabilities in human cancers (Lengauer et al., 1998),Lengauer, Kinzler and Vogelstein wrote: “Karyotypic studies have shownthat the majority of cancers have lost or gained chromosomes, andmolecular studies indicate that karyotypic data actually underestimatethe true extent of such changes. Losses of heterozygosity, that is,losses of a maternal or paternal allele in a tumor, are widespread andare often accompanied by a gain of the opposite allele. A tumor couldlose the maternal chromosome 8, for example, while duplicating thepaternal chromosome 8, leaving the cell with a normal chromosome 8karyotype but an abnormal chromosome 8 ‘allelotype’. The ‘average’cancer of the colon, breast, pancreas or prostate may lose 25% of itsalleles and it is not unusual for a tumor to have lost over half of itsalleles.” These observations have since been reinforced and extended toalmost all human cancers, including practically all carcinomas, innumerous reports (see (McGranahan et al., 2012) for review). It is nowunambiguously established that nearly all individual tumors exhibitmultiple losses of full chromosomes, entire chromosomal arms orsub-chromosomal regions of varying size. New algorithms are beingrapidly developed (e.g., Sathirapongsasuti et al., 2011) for thedetermination of the LOH profile in any given cell sample based on theexome sequence data. While statistical bias may at present question thevalidity of some interpretations (Teo et al., 2012), such algorithms arelikely to improve and replace most other methodologies for establishingLOH profiles which had been employed for this purpose in the pre-NGS era

Early LOH events can be detected in premalignant cells of the sametissue, but not in surrounding normal cells (Barrett et al., 1999). LOHis irreversible and events can only accumulate, so that tumorheterogeneity reflects the accumulation of losses throughout tumorprogression. While tumor subclones can develop which differ in later LOHevents, the existence of a minimal LOH signature that is shared bypremalignant cells, putative tumor stem cells and all tumor subclones ina given patient, is expected to be the rule. Branches stemming from this‘trunk’ LOH pattern would still create a limited set of partiallyoverlapping signatures which, together, cover all tumor cells in thesame patient

An inevitable outcome of gross LOH events is the concomitant loss of allother genes residing on the deleted chromosomal material, and thesenaturally include many genes encoding transmembrane proteins. Concerningtheir identity, a catalog of 3,702 different human cell surface proteins(the ‘surfaceome’) has been compiled (Da Cunha et al., 2009). Theexpression of □42% of surfaceome genes display broad tissue distributionwhile □85 genes are expressed by all tissues examined, which is thehallmark of housekeeping genes. These genes are candidates, thedifferent polymorphic variants of which may serve as targets for theiCARs and aCARs of the present invention

More recently, Bausch-Fluck et al. (Bausch-Fluck et al., 2015) appliedtheir Chemoproteomic Cell Surface Capture technology to identify acombined set of 1492 cell surface glycoproteins in 41 human cell types.A large fraction of the surfaceome is expected to be expressed by anygiven tumor, each exhibiting a distinctive profile. Genes encoding cellsurface proteins were found to be slightly enriched forsingle-nucleotide polymorphisms (SNPs) in their coding regions than allother genes (Da Cunha et al., 2009). Polymorphic in-frame insertions anddeletions, which are rarer, further contribute to the number of variantsand likely exert more robust structural effects on the polypeptideproducts than peptide sequence-altering (nonsynonymous) SNPs.Altogether, a typical genome contains 10,000 to 12,000 sites withnonsynonymous variants and 190-210 in-frame insertions/deletions(Abecasis et al., 2010; Auton et al., 2015). These variants are notevenly distributed throughout the genome as highly polymorphic genessuch as the HLA locus (http://www.ebi.ac.uk/imgt/hla/stats.html) orcertain G-protein-coupled receptor (GPCR) genes (Lee et al., 2003; Ranaet al., 2001) create distinct variant ‘hotspots’. Another layer ofLOH-related hotspots stems from the frequent loss of certainchromosomes, or chromosome arms in different cancers (e.g., 3p and 17pin small-cell lung carcinoma (Lindblad-Toh et al., 2000), 17p and 18q incolorectal cancer (Vogelstein et al., 1989), 17q and 19 in breast cancer(Li et al., 2014; Wang et al., 2004) 9p in melanoma (Stark and Hayward,2007), 10q in glioblastoma (Ohgaki et al., 2004) and more)

A significant fraction of allelic variations in surface proteins wouldaffect the extracellular portion of the respective gene products,potentially creating distinct allele-restricted epitopes which, inprinciple, can be recognized and distinguished from other variants byhighly-specific mAbs. It is well documented that mAbs can be isolatedthat discriminate between two variants of the same protein which differin a single amino acid only (see, for example, an early example of mAbsthat recognize point mutation products of the Ras oncogene withexquisite specificity (Carney et al., 1986)). Interestingly, it wasshown that two mAbs specific to a single amino acid interchange in aprotein epitope can use structurally distinct variable regions fromtheir heavy and light chain V gene pools (Stark and Caton, 1991).Recently, Skora et al. (Skora et al., 2015) reported the isolation ofpeptide-specific scFvs which can distinguish between HLA-I-boundneopeptides derived from mutated KRAS and EGFR proteins and their wildtype counterparts, differing in both cases in one amino acid

All taken together, a unique antigenic signature of tumor cells emerges,that can allow their unequivocal discrimination from all other cells inthe entire body of the individual patient. It comprises alltransmembrane proteins encoded by allelic variants that are absent fromthe tumor cell surface owing to LOH but are present on normal cells ofthe cancer tissue of origin or other tissues expressing these genes.Naturally, each gene affected by LOH will be characterized by a distinctpattern of tissue distribution except for true housekeeping genes. Themajority of these genes are not expected to be directly involved intumorigenesis or maintenance of the transformed phenotype and, in thissense, their loss is of a ‘passenger’ nature

The rationale presented above argues that a unique molecular portrayalis inevitably shaped by LOH for almost all tumors, which is marked bythe absence of numerous polymorphic surface structures that are presenton normal cells. Converting this postulated signature of the individualtumor to a targetable set of antigenic epitopes entails a practicableimmunological strategy for translating the recognition of a particular‘absence’ into an activating cue capable of triggering target cellkilling. Importantly, the incorporation of a safety device to assurethat on-target off-tumor reactivity is strictly avoided will be highlyfavorable in future clinical implementation of this strategy

The present invention tackles this challenge through the co-expressionin each therapeutic killer cell of a single pair of genes. One partnerin this pair encodes an activating CAR (aCAR) and the other encodes aprotecting CAR (pCAR) or an inhibitory CAR (iCAR)

II. Select Definitions

The term “nucleic acid molecule” as used herein refers to a DNA or RNAmolecule.

The term “encoding” refers to the inherent property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of other polymers andmacromolecules in biological processes having either a defined sequenceof nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence ofamino acids and the biological properties resulting therefrom. Thus, agene encodes a protein if transcription and translation of mRNAcorresponding to that gene produces the protein in a cell or otherbiological system. Both the coding strand, the nucleotide sequence ofwhich is identical to the mRNA sequence and is usually provided insequence listings, and the non-coding strand, used as the template fortranscription of a gene or cDNA, can be referred to as encoding theprotein or other product of that gene or cDNA.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence.Nucleotide sequences that encode proteins and RNA may include introns.

The term “endogenous” refers to any material from or produced inside anorganism, cell, tissue or system.

The term “exogenous” refers to any material introduced from or producedoutside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., lentiviruses, retroviruses, adenoviruses, andadeno-associated viruses) that incorporate the recombinantpolynucleotide.

The term “genomic variant” as used herein refers to a change of at leastone nucleotide at the genomic level in a sequenced sample compared tothe reference or consensus sequence at the same genomic position.

The term “corresponding reference allele” as used herein with referenceto a variant means the reference or consensus sequence or nucleotide atthe same genomic position as the variant.

The term “extracellular domain” as used herein with reference to aprotein means a region of the protein which is outside of the cellmembrane.

The term “loss of heterozygosity” or “LOH” as used herein means the lossof chromosomal materials such as a complete chromosome or a partthereof, in one copy of the two chromosomes in a somatic cell.

The term “sequence region” as used herein with reference to a variant ora reference allele means a sequence starting upstream and endingdownstream from the position of the variant, which can be translatedinto an “epitope peptide” that can be recognized by an antibody.

The term “CAR”, as that term is used herein, refers to a chimericpolypeptide that shares structural and functional properties with a cellimmune-function receptor or adaptor molecule, from e.g., a T cell or aNK cell. CARs include TCARs and NKR-CARs. Upon binding to cognateantigen, a CAR can activate or inactivate the cytotoxic cell in which itis disposed, or modulate the cell's antitumor activity or otherwisemodulate the cells immune response.

The term “specific binding” as used herein in the context of anextracellular domain, such as an scFv, that specifically binds to asingle allelic variant of a polymorphic cell surface epitope, refers tothe relative binding of the scFv to one allelic variant and its failureto bind to the corresponding different allelic variant of the samepolymorphic cell surface epitope. Since this depends on the avidity(number of CAR copies on the T cell, number of antigen molecules on thesurface of target cells (or cells to be protected) and the affinity ofthe specific CARs used, a functional definition would be that thespecific scFv would provide a significant signal in an ELISA against thesingle allelic variant of a polymorphic cell surface epitope to which itis specific or cells transfected with a CAR displaying the scFv would beclearly labeled with the single allelic variant of a polymorphic cellsurface epitope in a FACS assay, while the same assays using thecorresponding different allelic variant of the same polymorphic cellsurface epitope would not give any detectable signal.

The term “treating” as used herein refers to means of obtaining adesired physiological effect. The effect may be therapeutic in terms ofpartially or completely curing a disease and/or symptoms attributed tothe disease. The term refers to inhibiting the disease, e.g., arrestingits development; or ameliorating the disease, e.g., causing regressionof the disease.

As used herein, the terms “subject” or “individual” or “animal” or“patient” or “mammal,” refers to any subject, particularly a mammaliansubject, for whom diagnosis, prognosis, or therapy is desired, forexample, a human.

The phrase “safe effector immune cell” or “safe effector cell” includesthose cells described by the invention that express at least one iCAR orpCAR as described herein. In some embodiments, the “safe effector immunecell” or “safe effector cell” is capable of administration to a subject.In some embodiments, the “safe effector immune cell” or “safe effectorcell” further expresses an aCAR as described herein. In someembodiments, the “safe effector immune cell” or “safe effector cell”further expresses an iCAR or a pCAR as described herein. In someembodiments, the “safe effector immune cell” or “safe effector cell”further expresses an iCAR or a pCAR as described herein and an aCAR asdescribed herein.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. The carrier(s) mustbe “acceptable” in the sense of being compatible with the otheringredients of the composition and not deleterious to the recipientthereof.

The phrase “effective amount” or “therapeutically effective amount” areused interchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result.

The following exemplification of carriers, modes of administration,dosage forms, etc., are listed as known possibilities from which thecarriers, modes of administration, dosage forms, etc., may be selectedfor use with the present invention. Those of ordinary skill in the artwill understand, however, that any given formulation and mode ofadministration selected should first be tested to determine that itachieves the desired results.

Methods of administration include, but are not limited to, parenteral,e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal(e.g., oral, intranasal, buccal, vaginal, rectal, intraocular),intrathecal, topical and intradermal routes. Administration can besystemic or local. In some embodiments, the pharmaceutical compositionis adapted for oral administration.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the active agent is administered. The carriers in thepharmaceutical composition may comprise a binder, such asmicrocrystalline cellulose, polyvinylpyrrolidone (polyvidone orpovidone), gum tragacanth, gelatin, starch, lactose or lactosemonohydrate; a disintegrating agent, such as alginic acid, maize starchand the like; a lubricant or surfactant, such as magnesium stearate, orsodium lauryl sulphate; and a glidant, such as colloidal silicondioxide.

The term “peripheral blood mononuclear cell (PBMC)” as used hereinrefers to any blood cell having a round nucleus, such as a lymphocyte, amonocyte or a macrophage. Methods for isolating PBMCs from blood arereadily apparent to those skilled in the art. A non-limiting example isthe extraction of these cells from whole blood using ficoll, ahydrophilic polysaccharide that separates layers of blood, withmonocytes and lymphocytes forming a buffy coat under a layer of plasmaor by leukapheresis, the preparation of leukocyte concentrates with thereturn of red cells and leukocyte-poor plasma to the donor.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer, glioma, andthe like.

III. CAR-T System: iCARs, pCARs, and aCARs

It should be emphasized that the present invention provides a new avenueenabling specific targeting of tumor cells while keeping the normalcells secure. The concept presented herein provides for theidentification of new targets for iCARs (or pCARs or protective CARs),these targets defined as comprising single allelic variants ofpolymorphic cell surface epitopes, which are lost from tumor cells dueto LOH of the chromosomal region they reside in, while remainingexpressed on normal tissue. Because of the polymorphic variation, it ispossible to distinguish the two alleles and target only the allelemissing in the tumor cells. Further, the target antigen may notnecessarily itself be a tumor suppressor gene, or a gene predicted to beinvolved with cancer, since it is chosen for being in a region lost byLOH and could therefore simply be linked to such genes. This isconceptually different from the methods employed or suggested to date incancer therapy, which target tumor associated antigens or antigensdownregulated at tumors regardless of polymorphism. The present methodsalso provide for broadening the selection of aCAR beyond tumorassociated anitgens, by conferring protection of normal cells throughthe co-expression of the iCAR and/or pCAR as described herein.

The distinction is crucial because the LOH, being a genomic event,results in a total loss of a specific variant from the tumor with a veryrare probability of gaining back the lost allele. If the LOH eventoccurs very early in the development of tumors, it ensures a uniformtarget signature in all tumor cells derived from the initialpre-malignant tissue including metastatic tumors. Additionally, LOHoccurs in almost all types of cancer and this concept can therefore berelied upon as a universal tool for developing markers relevant to allthese cancer types. Since the LOH events are to some extent random, thepresent invention further provides for selection of personalized tumormarkers for each individual cancer patient, based on the specific LOHevents which took place in that patient. The tools relied upon toexecute this concept, the aCARs and the iCARs, are well-known and can beeasily prepared using methods well-known in the art as taught forexample, in WO 2015/142314 and in U.S. Pat. No. 9,745,368, bothincorporated by reference as if fully disclosed herein.

According to one strategy, the two CARs in every given pair specificallyrecognize the product of a different allelic variant of the same targetgene for which the patient is heterozygous. The basic principle is asfollows: the aCAR targets an allelic variant of a selected cell surfaceprotein that is expressed by the given tumor cells and is not affectedby LOH while the pCAR or iCAR targets the product encoded by the allelicvariant of the same gene that has been lost from these tumor cells dueto LOH. In other normal tissues of that individual patient that expressthe said gene, both alleles are present and are known to be equallyfunctional, that is, expression is biallelic in all tissues (in contrastto other genes which may exhibit random monoallelic expression (Chess,2012; Savova et al., 2016). In one scenario, the two CARs target tworelated epitopes residing at the same location on the protein product,which differ by one, or only few amino acids. In another scenario, theaCAR targets a non-polymorphic epitope on the same protein while thepCAR or iCAR is allele-specific. In this case the density of the aCARepitope on normal cells would generally be two-fold higher than that ofthe iCAR or pCAR one. In some embodiments, a single nucleic acid vectorencodes both the aCAR and iCAR or pCAR.

Another strategy utilizes as the pCAR or iCAR targets the proteinproducts of housekeeping genes. Since, by definition, these genes areexpressed on all cells in the body, they are safe targets for pCAR oriCARs. That is, if the pCAR or iCAR targets a membrane product of ahousekeeping gene for which the given patient is heterozygous, all cellsin the body, except the tumor cells which have lost this allele due toLOH, will be protected. This strategy allows for the uncoupling of theaCAR target gene product from the pCAR or iCAR one. In fact, the aCARtarget can then be any non-polymorphic epitope expressed by the tumor. Avariation of this strategy would be to utilize a known aCAR targeted toa non-polymorphic tumor-associated antigen, e.g., an aCAR in clinicaluse or under examination in clinical trials, in combination with an iCARor pCAR directed against a membrane product of a gene for which thegiven patient is heterozygous and which is expressed in at least thetissue of origin of the tumor and preferably in additional vital normaltissues in which aCAR target antigen is expressed.

Following the same rationale which allows the uncoupling of the aCARtarget antigen from the iCAR/pCAR one, the latter should not necessarilybe the product of a housekeeping gene. In some embodiments, the iCARand/or pCAR be the product of any gene the expression pattern of whichis sufficiently wide so as to protect vital normal tissues expressingthe aCAR target antigen in addition to the tumor. As a corollary, theaCAR antigen can be, as argued for housekeeping genes, anynon-polymorphic epitope expressed by the tumor, not restricted to known‘tumor-associated antigens’, a consideration which can vastly expand thelist of candidate aCAR targets. In general, for both housekeeping andnon-housekeeping genes, the identity of such normal vital tissues andlevel of expression would serve as important criteria in theprioritization of such candidate aCAR targets

Care must be taken to ensure that the inhibitory signal transmitted bythe iCAR is strictly and permanently dominant over the aCAR signal andthat no cross-recognition between the iCAR and the aCAR occurs.Dominance of the iCAR guarantees that activation of the killer cell uponencounter with normal cells expressing both alleles would be prevented.This default brake would, however, not operate upon engagement withtumor cells: in the absence of its target antigen the iCAR would notdeliver inhibitory signals, thus unleashing the anticipatedaCAR-mediated cellular activation and subsequent tumor cell lysis

The iCAR technology may be based on immune checkpoints. In this regard,the demonstration (Fedorov et al., 2013b; WO 2015/142314) that theregulatory elements of PD-1 and CTLA-4 possess a potent T cellinhibitory capacity when incorporated as iCAR signaling components isencouraging but the generality of these observations was recentlyquestioned (Chicaybam and Bonamino, 2014, 2015). Furthermore, althoughthe precise molecular pathways triggered by these checkpoint proteinsare not fully understood, their engagement dampens T-cell activationthrough both proximal and distal mechanisms, rendering T cellsunresponsive to concomitant activating stimuli (Nirschl and Drake,2013). Hence, although the inactivation status secured by PD-1 andCTLA-4 iCARs is indeed temporary and reversible (Fedorov et al., 2013b),it would not allow T cell activation in tissues expressing both iCAR andaCAR targets. In contrast, the dominance of NK inhibitory receptors overactivating receptors assures that healthy cells are spared from NK cellattack through a spatial, rather than temporal mechanism. (Long et al.,2013). There is compelling evidence that a single NK cell can spare aresistant cell expressing both inhibitory and activating ligands yet,kill a susceptible cell it simultaneously engages, which expresses onlythe activating ligands. This exquisite ability is governed by thedifferent spatial organization of signal transduction molecules formedat each of the respective immune synapses which consequently affects theexocytosis of cytolytic granules (e.g., Abeyweera et al., 2011; Erikssonet al., 1999; Treanor et al., 2006; Vyas et al., 2001; U.S. Pat. No.9,745,368).

The strategy based on the control asserted by iCARs depends on thedominance of the iCAR activity over the aCAR activity as explainedabove. In some embodiments, the present invention provides this type ofiCAR, termed here a pCAR (for ‘protective CAR, see FIG. 2), designed tooperate in CAR T cells in a synapse-selective manner and guarantee fulldominance over the co-expressed aCAR. In some embodiments, the iCARprovided by the present invention is this particular type of iCARreferred to herein as a protective CAR (pCAR).

In some embodiments, the pCAR of the present invention integrates twotechnological feats. First, the pCAR allows for uncoupling theactivating moiety of the aCAR (FcRγ/CD3-ζ) from the recognition unit andthe co-stimulatory element (e.g., CD28, 4-1BB, CD134 (OX40, GITR, IL2Rβand STAT3 binding motif (YXXQ)) by genetically placing them on twodifferent polypeptide products. Recoupling of these elements, which ismandatory for the aCAR function, will only take place by the addition ofa heterodimerizing drug which can bridge the respective binding sitesincorporated onto each of the polypeptides separately (FIG. 2B). Thereconstruction of a fully functional CAR by bridging similarly splitrecognition and activating moieties by virtue of a heterodimerizing drughas recently been reported by Wu et al. (Wu et al., 2015). For thispurpose, these authors used the FK506 binding protein domain (FKBP, 104amino acids) and the T2089L mutant of FKBP-rapamycin binding domain(FRB, 89 amino acids) that heterodimerize in the presence of therapamycin analog AP21967 (Scheme I below). This drug possess 1000-foldless immunosuppressive activity compared to rapamycin (Bayle et al.,2006; Graef et al., 1997; Liberles et al., 1997) and is commerciallyavailable (ARGENT™, Regulated Heterodimerization Kit, ARIAD). In someembodiments, the drug is administered orally.

Second, engrafting the pCAR recognition unit and the missing activatingdomain, respectively, onto the two surfaces of the transmembrane domainof a RIP-controlled receptor which contains the two intramembranecleavage sites (FIG. 2A). Binding of the pCAR to its antigen willtrigger dual cleavage of the encoded polypeptide first by a member ofthe extracellular disintegrin and metalloproteinase (ADAM) family whichremoves the ectodomain and then by intracellular γ-secretase, whichliberates the intracellular domain of the pCAR. This first cleavageevent is predicted to disrupt the ability of the truncated aCAR to gainaccess to a functional, membrane-anchored configuration of its missingactivating element, thus acquiring an operative mode (FIG. 2C). Thisprinciple was recently exploited in the development of new geneticswitches designed to limit CAR T cell activity to simultaneousrecognition of two different antigens on the tumor cell, applying eitherthe Notch receptor (Morsut et al., 2016; Roybal et al., 2016b) orEpithelial cell adhesion molecule (EpCAM, Pizem, Y., M.Sc. thesis underthe supervision of the Inventor), two well-studied receptors functioningthrough RIP. In these studies, binding of the RIP-based CAR to oneantigen releases a genetically-engineered intracellular domain whichtranslocates to the cell nucleus where it turns on the expression of thesecond CAR. Unlike the current invention which utilizes this processsolely for disarming any potential aCAR activity in the presence of theprotective antigen. In some embodiments, the first cleavage eventdisrupts the ability of the truncated aCAR to gain access to afunctional, membrane-anchored configuration of its missing activatingelement, thus acquiring an operative mode.

The proposed mode of action described above is predicted to exert localeffects so that only aCARs which reside in the same synapse are affectedand are no more able to bind their antigen productively and form animmunological synapse. As a result, even when multiple interactions ofthe aCAR with large numbers of non-tumor cells are likely to take place,they are only expected to be transient and nonfunctional so that thecells are fully capable of further interactions.

Dominance of the pCARs over their aCARs counterparts is inherent to thissystem as function of the aCARs utterly depends on presence of thepCARs. Relative shortage of pCARs in a given T cell would render theaCARs non-functional due to lack of an activating domain. In someembodiments, a shortage of pCARs in a given T cell renders the aCARsnon-functional due to lack of an activating domain.

It is critical that both the recognition domain and the activating oneare localized to the plasma membrane (Wu et al., 2015). Therefore, thesecond cleavage, which detaches the activating domain from the plasmamembrane, would render this domain nonfunctional and prevent unwantedcellular activation. In some embodiments, the recognition domain and theactivating one are localized to the plasma membrane. In someembodiments, the second cleavage detaches the activating domain from theplasma membrane and renders this domain nonfunctional and preventsunwanted cellular activation.

The aCAR and pCAR are designed to function via mutually exclusivemechanisms. The ability of the pCAR to undergo cleavage does not dependon the strength of inhibitory signaling so no completion on signalingoutcome will take place. As long as the pCARs are cleaved, the aCARscannot function, regardless of relative avidity of their interactionswith their respective antigens, a scenario which secures another cruciallevel of safety.

In some embodiments, the mammalian tissue is human tissue and in otherembodiments the related mammalian normal tissue is normal tissue fromwhich the tumor developed.

In some embodiments, the effector immune cell is a T cell, a naturalkiller cell or a cytokine-induced killer cell.

In some embodiments, the at least one signal transduction elementcapable of inhibiting an effector immune cell is homologous to a signaltransduction element of an immune checkpoint protein, such as an immunecheckpoint protein selected from the group consisting of PD1; CTLA4;BTLA; 2B4; CD160; CEACAM, such as CEACAM1; KIRs, such as KIR2DL1,KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2,KIR3DL3, LIR1, LIR2, LIR3, LIR5, LIR8 and CD94-NKG2A; LAG3; TIM3;V-domain Ig suppressor of T cell activation (VISTA); STimulator ofINterferon Genes (STING); immunoreceptor tyrosine-based inhibitory motif(ITIM)-containing proteins, T cell immunoglobulin and ITIM domain(TIGIT), and adenosine receptor (e.g., A2aR). In some embodiments, theimmune checkpoint protein is a negative immune regulator. In someembodiments, the negative immune regulator is selected from the groupconsisting of 2B4, LAG-3 and BTLA-4.

In some embodiments, immune checkpoint protein is a natural killer cellinhibitory receptor, e.g., KIRs, such as KIR2DL1, KIR2DL2, KIR2DL3,KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3; or a LeukocyteIg-like receptor, such as LIR1, LIR2, LIR3, LIR5, LIR8; and CD94-NKG2A,a C-type lectin receptor which forms heterodimers with CD94 and contains2 ITIMs.

The methods for preparing and using killer cell receptors in iCARs hasbeen described in U.S. Pat. No. 9,745,368, incorporated by reference asif fully disclosed herein.

In some embodiments, the extracellular domain of any one of the aboveembodiments is fused through a flexible hinge and transmembrane canonicmotif to said intracellular domain.

i. Target Identification: aCAR, iCAR and pCAR

The present invention provides methods for identification of aCAR, iCARand/or pCAR targets based identification of candidate genes havingextracellular polymorphic epitopes. In some embodiments, the aCAR can bedirected at any extracellular protein expressed on the tumor tissue. Insome embodiments, aCAR target is further expressed on non-tumor tissuesand the iCAR target is also expressed on non-tumor tissues but is notexpressed on tumor tissues.

In some embodiments, the method of identification of candidate genesincludes first determining that the gene encodes a transmembrane proteincomprising an extracellular polymorphic epitope. In some embodiments,the method of identification of candidate genes further includesdetermining that the gene has at least two expressed alleles. In someembodiments, these alleles exhibit at least one allelic variation. Insome embodiments, the allelic variation includes, for example, thepresence of one or more SNPs, insertions, and/or deletions. In someembodiments, the allelic variation found for the gene causes an aminoacid change relative to the reference sequence in an extracellularregion of the protein. In some embodiments, the gene is located in achromosomal region which undergoes loss of heterozygosity (LOH). In someembodiments, the gene is located in a chromosomal region which undergoesloss of heterozygosity (LOH) in cancer. In some embodiments, the gene isexpressed in a tissue-of-origin of a tumor type in which thecorresponding region was found to undergo LOH. In some embodiments, thegene is expressed at least in one or more tissues that the aCAR isexpressed in. In some embodiments, the iCAR or pCAR target is expressedin vital organ cells the aCAR is expressed in.

In some embodiments, the target for use in the iCAR and/or pCAR isselected based on identification of a gene having at least oneextracellular polymorphic epitope and wherein said gene has at least twoexpressed alleles. In some embodiments, the target for use in the iCARand/or pCAR is selected based on identification of a gene having locatedin a chromosomal region which undergoes loss of heterozygosity. In someembodiments, the target for use in the iCAR and/or pCAR is selectedbased on identification of a gene having located in a chromosomal regionwhich undergoes loss of heterozygosity in cancer. In some embodiments,the score for a theoretical SNP is calculated and a threshold limitdetermined. For example, if only 32% of the SNPs had a tumor suppressorgene on the chromosome, then the percentile rank for having one would be0.68. Further, for example, if the allele had a minor allele fraction of0.49 (where 0.5 is the highest possible), then the percentile rank wouldbe 0.99. If the rate of LOH was 0.10, and 75% of SNPs had more LOH thanthat, then the percentile rank would be 0.25. If the ratio of standarddeviation of expression values across tissues to the median for the geneharboring this SNP was 1.3 and that is better than 90% of other genes,then the percentile rank is 0.9. The total score for this SNP would thenbe 0.68*0.99*0.25*0.9=0.15. In some embodiments, this LOH candidatescore can be employed as one method for determining if a candidate geneis a suitable iCAR or pCAR target. In some embodiments, the target canbe selected based on this LOH score. In some embodiments, the candidategene is a determined to be suitable as an iCAR or pCAR target. LOHcandidates based on an LOH candidate score of greater than 0.4.

In some embodiments, the target for use in the iCAR and/or pCAR isselected from a gene having at least one extracellular polymorphicepitope. In some embodiments, the target is a gene is located onchromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5,chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10,chromosome 11, chromosome 12, chromosome 13, chromosome 14, chromosome15, chromosome 16, chromosome 17, chromosome 18, chromosome 19,chromosome 20, chromosome 21, chromosome 22, or chromosome X.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 1. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofABCA4, ADAM30, AQP10, ASTN1, C1orf101, CACNA1S, CATSPER4, CD101,CD164L2, CD1A, CD1C, CD244, CD34, CD46, CELSR2, CHRNB2, CLCA2, CLDN19,CLSTN1, CR1, CR2, CRB1, CSF3R, CSMD2, ECE1, ELTD1, EMC1, EPHA10, EPHA2,EPHA8, ERMAP, FCAMR, FCER1A, FCGR1B, FCGR2A, FCGR2B, FCGR3A, FCRL1,FCRL3, FCRL4, FCRL5, FCRL6, GJB4, GPA33, GPR157, GPR37L1, GPR88, HCRTR1,IGSF3, IGSF9, IL22RA1, IL23R, ITGA10, KIAA1324, KIAA2013, LDLRAD2, LEPR,LGR6, LRIG2, LRP8, LRRC52, LRRC8B, LRRN2, LY9, MIA3, MR1, MUC1, MXRA8,NCSTN, NFASC, NOTCH2, NPR1, NTRK1, OPN3, OR10J1, OR10J4, OR10K1, OR1OR2,OR10T2, OR10X1, OR11L1, OR14A16, OR14I1, OR14K1, OR2AK2, OR2C3, OR2G2,OR2G3, OR2L2, OR2M7, OR2T12, OR2T27, OR2T1, OR2T3, OR2T29, OR2T33,OR2T34, OR2T35, OR2T3, OR2T4, OR2T5, OR2T6, OR2T7, OR2T8, OR2W3, OR6F1,OR6K2, OR6K3, OR6K6, OR6N1, OR6P1, OR6Y1, PDPN, PEAR1, PIGR, PLXNA2,PTCH2, PTCHD2, PTGFRN, PTPRC, PTPRF, PTGFRN, PVRL4, RHBG, RXFP4, S1PR1,SCNN1D, SDC3, SELE, SELL, SELP, SEMA4A, SEMA6C, SLAMF7, SLAMF9, SLC2A7,SLC5A9, TACSTD2, TAS1R2, TIE1, TLR5, TMEM81, TNFRSF14, TNFRSF1B,TRABD2B, USH2A, VCAM1, and ZP4.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 2. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofABCG5, ALK, ASPRV1, ATRAID, CD207, CD8B, CHRNG, CLEC4F, CNTNAP5, CRIM1,CXCR1, DNER, DPP10, EDAR, EPCAM, GPR113, GPR148, GPR35, GPR39, GYPC,IL1RL1, ITGA4, ITGA6, ITGAV, LCT, LHCGR, LRP1B, LRP2, LY75, MARCO,MERTK, NRP2, OR6B2, PLA2R1, PLB1, PROKR1, PROM2, SCN7A, SDC1, SLC23A3,SLC5A6, TGOLN2, THSD7B, TM4SF20, TMEFF2, TMEM178A, TPO, and TRABD2A.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 3. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofACKR2, ALCAM, ANO10, ATP13A4, BTLA, CACNA1D, CACNA2D2, CACNA2D3, CASR,CCRL2, CD200, CD200R1, CD86, CD96, CDCP1, CDHR4, CELSR3, CHL1, CLDN11,CLDN18, CLSTN2, CSPG5, CX3CR1, CXCR6, CYP8B1, DCBLD2, DRD3, EPHA6,EPHB3, GABRR3, GP5, GPR128, GPR15, GPR27, GRM2, GRM7, HEG1, HTR3C,HTR3D, HTR3E, IGSF11, IL17RC, IL17RD, IL17RE, IL5RA, IMPG2, ITGA9,ITGB5, KCNMB3, LRIG1, LRRC15, LRRN1, MST1R, NAALADL2, NRROS, OR5AC1,OR5H1, OR5H14, OR5H15, OR5H6, OR5K2, OR5K3, OR5K4, PIGX, PLXNB1, PLXND1,PRRT3, PTPRG, ROBO2, RYK, SEMA5B, SIDT1, SLC22A14, SLC33A1, SLC4A7,SLITRK3, STAB1, SUSD5, TFRC, TLR9, TMEM108, TMEM44, TMPRSS7, TNFSF10,UPK1B, VIPR1, and ZPLD1.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 4. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofANTXR2, BTC, CNGA1, CORIN, EGF, EMCN, ENPEP, EPHA5, ERVMER34-1, EVC2,FAT1, FAT4, FGFRL1, FRAS1, GPR125, GRID2, GYPA, GYPB, KDR, KIAA0922,KLB, MFSD8, PARM1, PDGFRA, RNF150, TENM3, TLR10, TLR1, TLR6, TMEM156,TMPRSS11A, TMPRSS11B, TMPRSS11E, TMPRSS11F, UGT2A1, and UNC5C.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 5. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofADAM19, ADRB2, BTNL3, BTNL8, BTNL9, C5orf15, CATSPER3, CD180, CDH12,CDHR2, COL23A1, CSF1R, F2RL2, FAM174A, FAT2, FGFR4, FLT4, GABRA6,GABRG2, GPR151, GPR98, GRM6, HAVCR1, HAVCR2, IL31RA, IL6ST, IL7R,IQGAP2, ITGA1, ITGA2, KCNMB1, LIFR, LNPEP, MEGF10, NIPAL4, NPR3, NRG2,OR2V1, OR2Y1, OSMR, PCDH12, PCDH1, PCDHA1, PCDHA2, PCDHA4, PCDHA8,PCDHA9, PCDHB10, PCDHB11, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB2,PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHGA1, PCDHGA4, PDGFRB, PRLR, SEMA5A,SEMA6A, SGCD, SLC1A3, SLC22A4, SLC22A5, SLC23A1, SLC36A3, SLC45A2,SLC6A18, SLC6A19, SLCO6A1, SV2C, TENM2, TIMD4, and UGT3A1.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 6. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofBAI3, BTN1A1, BTN2A1, BTN2A2, BTN3A1, BTN3A2, BTNL2, CD83, DCBLD1, DLL1,DPCR1, ENPP1, ENPP3, ENPP4, EPHA7, GABBR1, GABRR1, GCNT6, GFRAL, GJB7,GLP1R, GPR110, GPR111, GPR116, GPR126, GPR63, GPRC6A, HFE, HLA-A, HLA-B,HLA-C, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1,HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-G, IL20RA, ITPR3,KIAA0319, LMBRD1, LRFN2, LRP11, MAS1L, MEP1A, MICA, MICB, MOG, MUC21,MUC22, NCR2, NOTCH4, OPRM1, OR10C1, OR12D2, OR12D3, OR14J1, OR2B2,OR2B6, OR2J1, OR2W1, OR5V1, PDE10A, PI16, PKHD1, PTCRA, PTK7, RAET1E,RAET1G, ROS1, SDIM1, SLC16A10, SLC22A1, SLC44A4, TAAR2, TREM1, TREML1,and TREML2. In some embodiments, the gene comprising the extracellularpolymorphic epitope is located on chromosome 6 and comprises an HLAtarget. In some embodiments, the target for use in the iCAR and/or pCARis HLA-A, HLA-B, HLA-C, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2,HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-G. In someembodiments, the target for use in the iCAR and/or pCAR is HLA-A2,

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 7. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofAQP1, C7orf50, CD36, CDHR3, CNTNAP2, DPP6, EGFR, EPHA1, EPHB6, ERVW-1,GHRHR, GJC3, GPNMB, GRM8, HUS1, HYAL4, KIAA1324L, LRRN3, MET, MUC12,MUC17, NPC1L1, NPSR1, OR2A12, OR2A14, OR2A25, OR2A42, OR2A7, OR2A2,OR2AE1, OR2F2, OR6V1, PILRA, PILRB, PKD1L1, PLXNA4, PODXL, PTPRN2,PTPRZ1, RAMP3, SLC29A4, SMO, TAS2R16, TAS2R40, TAS2R4, TFR2, THSD7A,TMEM213, TTYH3, ZAN, and ZP3.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 8. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofADAM18, ADAM28, ADAM32, ADAM7, ADAMS, ADRA1A, CDH17, CHRNA2, CSMD1,CSMD3, DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1, PRSS55, SCARA3,SCARA5, SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A1, TNFRSF10A, andTNFRSF10B.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 9. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofABCA1, AQP7, ASTN2, C9orf135, CA9, CD72, CNTNAP3B, CNTNAP3, CRB2,ENTPD8, GPR144, GRIN3A, IZUMO3, KIAA1161, MAMDC4, MEGF9, MUSK, NOTCH1,OR13C2, OR13C3, OR13C5, OR13C8, OR13C9, OR13D1, OR13F1, OR1B1, OR1J2,OR1K1, OR1L1, OR1L3, OR1L6, OR1L8, OR1N1, OR1N2, OR1Q1, OR2S2, PCSK5,PDCD1LG2, PLGRKT, PTPRD, ROR2, SEMA4D, SLC31A1, TEK, TLR4, TMEM2, andVLDLR.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 10. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofABCC2, ADAM8, ADRB1, ANTXRL, ATRNL1, C10orf54, CDH23, CDHR1, CNNM2,COL13A1, COL17A1, ENTPD1, FZD8, FGFR2, GPR158, GRID1, IL15RA, IL2RA,ITGA8, ITGB1, MRC1, NRG3, NPFFR1, NRP1, OPN4, PCDH15, PKD2L1, PLXDC2,PRLHR, RET, RGR, SLC16A9, SLC29A3, SLC39A12, TACR2, TCTN3, TSPAN15,UNC5B, and VSTM4.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 11. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofAMICA1, ANO1, ANO3, APLP2, C11orf24, CCKBR, CD248, CD44, CD5, CD6, CD82,CDON, CLMP, CRTAM, DCHS1, DSCAML1, FAT3, FOLH1, GDPD4, GDPD5, GRIK4,HEPHL1, HTR3B, IFITM10, IL10RA, KIRREL3, LGR4, LRP4, LRP5, LRRC32, MCAM,MFRP, MMP26, MPEG1, MRGPRE, MRGPRF, MRGPRX2, MRGPRX3, MRGPRX4, MS4A4A,MS4A6A, MTNR1B, MUC15, NAALAD2, NAALADL1, NCAM1, NRXN2, OR10A2, OR10A5,OR10A6, OR10D3, OR10G4, OR10G7, OR10G8, OR10G9, OR10Q1, OR10S1, OR1S1,OR2AG1, OR2AG2, OR2D2, OR4A47, OR4A15, OR4A5, OR4C11, OR4C13, OR4C15,OR4C16, OR4C3, OR4C46, OR4C5, OR4D6, OR4A8P, OR4D9, OR4S2, OR4X1,OR51E1, OR51L1, OR52A1, OR52E1, OR52E2, OR52E4, OR52E6, OR52I1, OR52I2,OR52J3, OR52L1, OR52N1, OR52N2, OR52N4, OR52W1, OR56B1, OR56B4, OR5A1,OR5A2, OR5AK2, OR5AR1, OR5B17, OR5B3, OR5D14, OR5D16, OR5D18, OR5F1,OR5I1, OR5L2, OR5M11, OR5M3, OR5P2, OR5R1, OR5T2, OR5T3, OR5W2, OR6A2,OR6T1, OR6X1, OR8A1, OR8B12, OR8B2, OR8B3, OR8B4, OR8D1, OR8D2, OR8H1,OR8H2, OR8H3, OR8I2, OR8J1, OR8J2, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1,OR9G1, OR9G4, OR9Q2, P2RX3, PTPRJ, ROBO3, SIGIRR, SLC22A10, SLC3A2,SLC5A12, SLCO2B1, SORL1, ST14, SYT8, TENM4, TMEM123, TMEM225, TMPRSS4,TMPRSS5, TRIM5, TRPM5, TSPAN18, and ZP1.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 12. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofANO4, AVPR1A, BCL2L14, CACNA2D4, CD163, CD163L1, CD27, CD4, CLEC12A,CLEC1B, CLEC2A, CLEC4C, CLEC7A, CLECL1, CLSTN3, GPR133, GPRC5D, ITGA7,ITGB7, KLRB1, KLRC2, KLRC3, KLRC4, KLRF1, KLRF2, LRP1, LRP6, MANSC1,MANSC4, OLR1, OR10AD1, OR10P1, OR2AP1, OR6C1, OR6C2, OR6C3, OR6C4,OR6C6, OR6C74, OR6C76, OR8S1, OR9K2, ORAI1, P2RX4, P2RX7, PRR4, PTPRB,PTPRQ, PTPRR, SCNN1A, SELPLG, SLC2A14, SLC38A4, SLC5A8, SLC6A15, SLC8B1,SLCO1A2, SLCO1B1, SLCO1B7, SLCO1C1, SSPN, STAB2, TAS2R10, TAS2R13,TAS2R14, TAS2R20, TAS2R30, TAS2R31, TAS2R42, TAS2R43, TAS2R46, TAS2R7,TMEM119, TMEM132B, TMEM132C, TMEM132D, TMPRSS12, TNFRSF1A, TSPAN8, andVSIG10.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 13. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofATP4B, ATP7B, FLT3, FREM2, HTR2A, KL, PCDH8, RXFP2, SGCG, SHISA2,SLC15A1, SLITRK6, and TNFRSF19.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 14. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofADAM21, BDKRB2, C14orf37, CLEC14A, DLK1, FLRT2, GPR135, GPR137C, JAG2,LTB4R2, MMP14, OR11G2, OR11H12, OR11H6, OR4K1, OR4K15, OR4K5, OR4L1,OR4N2, OR4N5, SLC24A4, and SYNDIG1L.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 15. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofANPEP, CD276, CHRNA7, CHRNB4, CSPG4, DUOX1, DUOX2, FAM174B, GLDN,IGDCC4, ITGA11, LCTL, LTK, LYSMD4, MEGF11, NOX5, NRG4, OCA2, OR4F4,OR4M2, OR4N4, PRTG, RHCG, SCAMPS, SEMA4B, SEMA6D, SLC24A1, SLC24A5,SLC28A1, SPG11, STRA6, TRPM1, and TYRO3.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 16. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofATP2C2, CACNA1H, CD19, CDH11, CDH15, CDH16, CDH3, CDH5, CNGB1, CNTNAP4,GDPD3, GPR56, GPR97, IFT140, IL4R, ITFG3, ITGAL, ITGAM, ITGAX, KCNG4,MMP15, MSLNL, NOMO1, NOMO3, OR2C1, PIEZO1, PKD1, PKD1L2, QPRT, SCNN1B,SEZ6L2, SLC22A31, SLC5A11, SLC7A6, SPN, TMC5, TMC7, TMEM204, TMEM219,and TMEM8A.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 17. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofABCC3, ACE, AOC3, ARL17B, ASGR2, C17orf80, CD300A, CD300C, CD300E,CD300LF, CD300LG, CHRNB1, CLEC10A, CNTNAP1, CPD, CXCL16, ERBB2,FAM171A2, GCGR, GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3, ITGAE,ITGB3, KCNJ12, LRRC37A2, LRRC37A3, LRRC37A, LRRC37B, MRC2, NGFR, OR1A2,OR1D2, OR1G1, OR3A1, OR3A2, OR4D1, OR4D2, RNF43, SCARF1, SCN4A, SDK2,SECTM1, SEZ6, SHPK, SLC26A11, SLC5A10, SPACA3, TMEM102, TMEM132E,TNFSF12, TRPV3, TTYH2, and TUSC5.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 18. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofAPCDD1, CDH19, CDH20, CDH7, COLEC12, DCC, DSC1, DSG1, DSG3, DYNAP,MEP1B, PTPRM, SIGLEC15, and TNFRSF11A.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 19. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofABCA7, ACPT, BCAM, C19orf38, C19orf59, C5AR1, CATSPERD, CATSPERG, CD22,CD320, CD33, CD97, CEACAM19, CEACAM1, CEACAM21, CEACAM3, CEACAM4,CLEC4M, DLL3, EMR1, EMR2, EMR3, ERVV-1, ERVV-2, FAM187B, FCAR, FFAR3,FPR1, FXYD5, GFY, GP6, GPR42, GRIN3B, ICAM3, IGFLR1, IL12RB1, IL27RA,KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KIRREL2, KISS1R,LAIR1, LDLR, LILRA1, LILRA2, LILRA4, LILRA6, LILRB1, LILRB2, LILRB3,LILRB4, LILRB5, LINGO3, LPHN1, LRP3, MADCAM1, MAG, MEGF8, MUC16, NCR1,NOTCH3, NPHS1, OR10H1, OR10H2, OR10H3, OR10H4, OR1I1, OR2Z1, OR7A10,OR7C1, OR7D4, OR7E24, OR7G1, OR7G2, OR7G3, PLVAP, PTGIR, PTPRH, PTPRS,PVR, SCN1B, SHISA7, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC5, SIGLEC6,SIGLEC8, SIGLEC9, SLC44A2, SLC5A5, SLC7A9, SPINT2, TARM1, TGFBR3L, TMC4,TMEM91, TMEM161A, TMPRSS9, TNFSF14, TNFSF9, TRPM4, VN1R2, VSIG10L,VSTM2B, and ZNRF4.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 20. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofABHD12, ADAM33, ADRA1D, APMAP, ATRN, CD40, CD93, CDH22, CDH26, CDH4,FLRT3, GCNT7, GGT7, JAG1, LRRN4, NPBWR2, OCSTAMP, PTPRA, PTPRT, SEL1L2,SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3, SLC2A10, SLC4A11, SSTR4, andTHBD.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 21. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofCLDN8, DSCAM, ICOSLG, IFNAR1, IFNGR2, IGSF5, ITGB2, KCNJ15, NCAM2,SLC19A1, TMPRSS15, TMPRSS2, TMPRSS3, TRPM2, and UMODL1.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome 22. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofCACNA1I, CELSR1, COMT, CSF2RB, GGT1, GGT5, IL2RB, KREMEN1, MCHR1,OR11H1, P2RX6, PKDREJ, PLXNB2, SCARF2, SEZ6L, SSTR3, SUSD2, TMPRSS6, andTNFRSF13C.

In some embodiments, the gene comprising the extracellular polymorphicepitope is located on chromosome X. In some embodiments, the target foruse in the iCAR and/or pCAR is selected from the group consisting ofATP6AP2, ATP7A, CNGA2, EDA2R, FMR1NB, GLRA4, GPR112, GUCY2F, HEPH,P2RY10, P2RY4, PLXNA3, PLXNB3, TLR8, VSIG4, and XG.

In some embodiments, the aCAR used to treat the cancer is directedagainst or specifically binds to any membrane protein which is expressedon the tumor tissue as long as the iCAR is expressed on every normaltissue in which the targeted protein is expressed. In some embodiments,the aCAR can specifically bind or be directed to a tumor associatedprotein, tumor associated antigen and/or antigens in clinical trials, aCAR target as listed in Table 1, as well as any cell surface proteinthat is expressed in a tumor tissue to which an iCAR can be matched orpaired with regard to target binding, according to the criteria listedin the application. In some embodiments, the aCAR can be any expressedprotein with an extracellular domain, as long as the iCAR is expressedin the same tissues as the aCAR or in any vital tissues, but is lost inthe tumor cells. In some embodiments, the aCAR used to treat the cancer,such as any one of the cancer types recited above, is directed againstor specifically binds to, a non-polymorphic cell surface epitopeselected from the antigens listed in Table 1, such as CD19. In someembodiments, the aCAR, iCAR, and/or pCAR target is any target with anextracellular domain. In some embodiments, the aCAR used to treat thecancer, is directed against or specifically binds to, a non-polymorphiccell surface epitope selected from but not limited to the following listof antigens: CD19, CD20, CD22, CD10, CD7, CD49f, CD56, CD74, CAIX Igκ,ROR1, ROR2, CD30, LewisY, CD33, CD34, CD38, CD123, CD28, CD44v6, CD44,CD41, CD133, CD138, NKG2D-L, CD139, BCMA, GD2, GD3, hTERT, FBP, EGP-2,EGP-40, FR-α, L1-CAM, ErbB2,3,4, EGFRvIII, VEGFR-2, IL-13Rα2, FAP,Mesothelin, c-MET, PSMA, CEA, kRas, MAGE-A1, MUC1MUC16, PDL1, PSCA,EpCAM, FSHR, AFP, AXL, CD80 CD89, CDH17, CLD18, GPC3, TEM8, TGFB1,NY-ESO-1 WT-1 and EGFR. In some embodiments, the aCAR, iCAR, and/or pCARtarget is an antigen listed in Table 1.

TABLE 1 CAR target antigens, including some evaluated in trialsregistered in ClinicalTrials.gov Antigen Key structural/functionalfeatures Malignancy Potential off-tumor targets Hematologic CD19 Pan-Bcell marker involved in signal ALL, CLL, NHL, normal B cellsmalignancies transduction by the BCR HL, PLL CD20 Tetra-transmembrane,regulation of CLL, NHL normal B cells Ca transport and B-cell activationCD22 B-lineage specific adhesion receptor, ALL, NHL normal B cellssialic acid-binding Ig-type lectin family Igκ Ig light chain isotypeexpressed by CLL, NHL, MM normal B cells approx. 65% of normal human Bcells ROR1 Type I orphan-receptor tyrosine- CLL, NHL pancreas; adiposecells kinase-like, survival-signaling receptor in tumors CD30 TNFRmember, pleiotropic effects on NHL, TCL, HL resting CD8 T cells;activated B cell growth and survival involving and Th2 cells NF-κBLewis^(Y) (CD174) a membrane AML, MM early myeloid progenitor cellsoligosaccharide harboring two fucose groups CD33 Sialic acid-bindingIg-type lectin AML hematopoietic progenitors; serving as adhesionmolecule of the myelo-monocytic precursors; myelomonocytic lineagemonocytes CD123 The α chain of the IL-3 receptor AML BM myeloidprogenitors; DCs, B cells; mast cells, monocytes; macro-phages;megakar.; endothelial cells NKG2D-L Ligands for the NK and T-cell AML,MM gastrointestinal epithelium, activating receptor NKG2D, bearingendothelial cells and fibroblasts; similarity to MHC-I molecules;upregulated during inflammation CD139 Syndecan-1, cell surface heparanMM precursor & plasma B cells; sulfate proteoglycan, ECM receptorepithelia BCMA TNFR member, binds BAFF and MM B cells APRIL, involved inproliferation signaling TACI MM Mono-nuclear cells, heart Solid tumorsGD2 Disialoganglioside NB; sarcomas; solid skin; neurons tumors FR-αGPI-linked folate receptor, functions ovarian cancer apical surface inkidney, lung, in the uptake of reduced folate thyroid, kidney & breastcofactors epithelia L1-CAM CD171, neuronal cell adhesion NB CNS;sympathetic ganglia; molecule of the Ig superfamily adrenal medullaErbB2 HER2, Member of the EGFR family brain, CNS, glioma,gastrointestinal, respiratory, of receptor tyrosine-protein kinases GBM,H&N, solid reproductive & urinary tracts tumors epithelia, skin, breast& placenta; hematopoietic cells EGFRvIII Splice variant, in-framedeletion in brain, CNS, gliomas, none the amplified EGFR gene encoding aGBM truncated extracellular domain that constantly delivers pro-survivalsignals VEGFR-2 type III transmembrane kinase solid tumors vascular andlymphatic receptor of the Ig superfamily, endothelia regulates vascularendothelial function IL-13Rα2 The α chain of one of the two IL-13 brain,CNS, gliomas, astrocytes; brain; H&N tissue receptors GBM FAP Cellsurface serine protease Mesothelioma fibroblasts in chronicinflammation, wound healing, tissue remodeling Mesothelin 40-kDa cellsurface glycoprotein mesothelioma, peritoneal, pleural, and with unknownfunction pancreatic, ovarian pericardial mesothelial surfaces c-METhepatocyte growth factor receptor TNBC liver, gastrointestinal tract,(HGFR), disulfide linked α-β thyroid, kidney, brain heterodimericreceptor tyrosine kinase PSMA type II membrane glycoprotein Prostateapical surface of normal possessing N-Acetylated alpha- prostate andintestinal linked acidic dipeptidase and folate epithelium and renalproximal hydrolase activity tubular cells CEA surface glycoprotein,member of the colorectal, breast, apical epithelial surface: colon, Igsuperfamily and of the CEA- solid tumors stomach, esophagus & tonguerelated family of cell adhesion molecules EGFR ErbB1, Her1, receptortyrosine Solid tumors tissues of epithelial, kinases signaling celldifferentiation mesenchymal & neuronal origin and proliferation uponligand binding 5T4 tumor-associated antigen which is Solid tumorstissues of epithelial origin expressed on the cell surface of m GPC3heparan sulfate proteoglycan, Solid tumors Urine tissue ROR1 ReceptorTyrosine Kinase Like Solid tumors as well Urine, pancrease, colon,ovary, Orphan Receptor as CLL brain, monocytes MUC genes O-glycosylatedprotein that play an Solid tumors Colon, kidney, lung, breast, (MUC-1,MUC- essential role in forming protective pancrease urine 16) mucousbarriers on epithelial surfaces PDL 1 an immune inhibitory receptorligand Lung Spleen, breast that is expressed by hematopoietic andnon-hematopoietic cells

TABLE 2 Other CAR target antigens Antigen Key structural/functionalfeatures Malignancy Hem. Malig. CD38 a surface cyclic ADP ribosehydrolase CLL, NHL, MM involved in transmembrane signaling and celladhesion CS1 Cell surface signaling lymphocytic MM activation molecule(SLAM) Solid tumors PSCA GPI-anchored membrane glycoprotein of prostate,bladder, pancreatic the Thy-1/Ly-6 family CD44v6 alternatively splicedvariant 6 of the H&N, liver, pancreatic, gastric, hyaluronate receptorCD44 breast, colon; AML, NHL, MM CD44v7/8 alternatively spliced variant7/8 of the breast, cervical hyaluronate receptor CD44 MUC1 denselyglycosylated member of the colon, lung, pancreas, breast, mucin familyof glycoproteins ovarian, prostate, kidney, stomach, H&N L-11rα the αsubunit of the IL-11 recepto colon, gastric, breast, prostate;osteosarcoma EphA2 erythropoietin-producing hepatocellular Glioma;breast, colon, ovarian, carcinoma A2 (EphA2) receptor, a prostate,pancreatic member of the Eph family of receptor tyrosine kinases CAIXtransmembrane zinc metalloenzyme RCC; tumors under hypoxia CSPG4 highmolecular weight melanoma- RCC; tumors under hypoxia associated antigen,cell surface proteoglycan

ii. Recognition Moiety: aCAR, iCAR and pCAR

The present invention also provides for recognition moieties designed toprovide specific binding to the target. The recognition moiety allowsfor directing the specific and targeted binding of the aCAR, iCAR and/orpCAR. In some embodiments, the recognition moiety designed to providespecific binding to the target provides specific binding to anextracellular polymorphic epitope. In some embodiments, the recognitionmoiety is part of an extracellular domain of the aCAR, iCAR and/or pCAR.In some embodiments, the extracellular domain comprises an antibody,derivative or fragment thereof, such as a humanized antibody; a humanantibody; a functional fragment of an antibody; a single-domainantibody, such as a Nanobody; a recombinant antibody; and a single chainvariable fragment (ScFv). In some embodiments, the extracellular domaincomprises an antibody mimetic, such as an affibody molecule; an affilin;an affimer; an affitin; an alphabody; an anticalin; an avimer; a DARPin;a fynomer; a Kunitz domain peptide; and a monobody. In some embodiments,the extracellular domain comprises an aptamer.

Generally, any relevant technology may be used to engineer a recognitionmoiety that confers to the aCARs and pCAR or iCARs specific binding totheir targets. For example, recognition moieties comprising thisiCAR-aCAR Library may be derived from a master recognition moiety poolideally selected from a combinatorial display library, so that:

-   -   Collectively, the selected recognition moieties target the        cell-surface products of an array of genes which reside on each        of the two arms of all 22 human autosomes. The shorter the        distance between neighboring genes the fuller the coverage        hence, the greater the universality of use.    -   For each of the selected genes a set of allele-specific        recognition moieties is isolated, each allowing rigorous        discrimination between different allelic variants that are        prevalent in the human population. The greater the number of        targeted variants, the greater the number of therapeutic gene        pairs that can be offered to patients.

A given allelic product can become a potential pCAR or iCAR target inone patient and a useful aCAR target in another patient harboring thesame allele, depending on the particular LOH pattern in each case.Hence, as suitable recognition moiety genes are identified, each will beengrafted onto both a pCAR or an iCAR and an aCAR gene scaffold. It istherefore desirable that all recognition moieties directed at allelicvariants of the same gene possess binding affinities of a similar range.Within such a given set of recognition moieties, all possiblecombinations of pCAR-aCAR or iCAR-aCAR pairs can be pre-assembled so asto assure the highest coverage of potential allelic compositions of thatgene in the entire population.

In some embodiments, the patient is heterozygous for the major alleleand a minor one, the products of which differ in a single position alongthe encoded polypeptide as a result of a nonsynonymous SNP or, lessfrequently, an indel. In some other embodiments, a patient isheterozygous for two minor alleles which differ from the major one intwo separate positions. Depending on the particular LOH event involvingthe said gene in individual patients, a given variant epitope can serveas an iCAR target in one patient and an aCAR target in another. In someembodiments, the variant epitope that can serve as an iCAR target is notthe major allele variant. In some embodiments, the variant epitope thatcan serve as the iCAR target is a minor allele.

The identification of a variant-specific mAb (say, a mAb specific to theepitope encoded by the minor allele ‘a’) is well known in the art and issimilar, in principle, to the identification of a mAb against anyconventional antigenic determinant, and can usually best be accomplishedvia high throughput screening of a recombinant antibody scFv library,utilizing, for example, phage (Barbas et al., 2004), ribosome (Hanes etal., 1997) or yeast (Chao et al., 2006) display technologies. Theantigen employed for library screening can either be a synthetic peptidespanning the position of variation between the two alleles (typically15-20 amino acid in length or more), a recombinant full-lengthpolypeptide which can either be commercially available ortailor-synthesized by one of the many companies operating in this field,or even entire cells expressing the said allelic variant at high levelby virtue of gene transfection (e.g., electroporation of mRNA encodingthe full-length cDNA cloned as template for in-vitro mRNA transcriptionin the pGEM4Z/A64 vector (Boczkowski et al., 2000)), following asubtraction step performed on the same cells not expressing this allele.These methods are well-known and described in e.g., Molecular Cloning: ALaboratory Manual (Fourth Edition) Green and Sambrook, Cold SpringHarbor Laboratory Press; Antibodies: A Laboratory Manual (SecondEdition), Edited by Edward A. Greenfield, 2012 CSH laboratory press;Using Antibodies, A laboratory manual by Ed Harlow and David Lane, 1999CSH laboratory press.

By definition, the corresponding epitope (at the same position) which isencoded by the major allele (‘A’), creates a unique antigenicdeterminant that differs from that created by ‘a’ in the identity of asingle amino acid (SNP) or length (indel; for example, insertion ordeletion). This determinant can, in principle, be recognized by adifferent set of mAbs identified by the same, or other, antibody displayscreening technology. The ability of distinct members in each of the twosets of identified mAbs to distinguish between the two epitopes orvariants, for example, an antibody from the first set binds the productof allele ‘a’ but not of ‘A’ and an Ab from the second set reciprocallybinds ‘A’ but not ‘a’ can be determined using conventional bindingassays such as ELISA or flow cytometry (Skora et al., 2015) or othertechnique for cell staining. Alternatively, once an ‘a’-binding Ab isidentified which does not bind ‘A’ and its protein sequence isdetermined, a computational method can potentially be used to predictthe sequence of a ‘complementary’ antibody scFv which binds ‘A’ but not‘a’. For such a computational method see, for example (Sela-Culang etal., 2015a,b).

In some embodiments, for example with regard to the HLA-class I locusgenes HLA-A, HLA-B, and HLA-C as the target genes, there are numerousallele-specific monoclonal antibodies available, for example, but notlimited to, the antibodies listed in Example 3.

In some embodiments, the target for use in generation of a recognitionmoiety comprises at least one extracellular polymorphic epitope. In someembodiments, the target is the product of a gene that is located onchromosome 1, chromosome 2, chromosome 3, chromosome 4, chromosome 5,chromosome 6, chromosome 7, chromosome 8, chromosome 9, chromosome 10,chromosome 11, chromosome 12, chromosome 13, chromosome 14, chromosome15, chromosome 16, chromosome 17, chromosome 18, chromosome 19,chromosome 20, chromosome 21, chromosome 22, or chromosome X.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 1. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ABCA4, ADAM30,AQP10, ASTN1, C1orf101, CACNA1S, CATSPER4, CD101, CD164L2, CD1A, CD1C,CD244, CD34, CD46, CELSR2, CHRNB2, CLCA2, CLDN19, CLSTN1, CR1, CR2,CRB1, CSF3R, CSMD2, ECE1, ELTD1, EMC1, EPHA10, EPHA2, EPHA8, ERMAP,FCAMR, FCER1A, FCGR1B, FCGR2A, FCGR2B, FCGR3A, FCRL1, FCRL3, FCRL4,FCRL5, FCRL6, GJB4, GPA33, GPR157, GPR37L1, GPR88, HCRTR1, IGSF3, IGSF9,IL22RA1, IL23R, ITGA10, KIAA1324, KIAA2013, LDLRAD2, LEPR, LGR6, LRIG2,LRP8, LRRC52, LRRC8B, LRRN2, LY9, MIA3, MR1, MUC1, MXRA8, NCSTN, NFASC,NOTCH2, NPR1, NTRK1, OPN3, OR10J1, OR10J4, OR10K1, OR1OR2, OR10T2,OR10X1, OR11L1, OR14A16, OR14I1, OR14K1, OR2AK2, OR2C3, OR2G2, OR2G3,OR2L2, OR2M7, OR2T12, OR2T27, OR2T1, OR2T3, OR2T29, OR2T33, OR2T34,OR2T35, OR2T3, OR2T4, OR2T5, OR2T6, OR2T7, OR2T8, OR2W3, OR6F1, OR6K2,OR6K3, OR6K6, OR6N1, OR6P1, OR6Y1, PDPN, PEAR1, PIGR, PLXNA2, PTCH2,PTCHD2, PTGFRN, PTPRC, PTPRF, PTGFRN, PVRL4, RHBG, RXFP4, S1PR1, SCNN1D,SDC3, SELE, SELL, SELP, SEMA4A, SEMA6C, SLAMF7, SLAMF9, SLC2A7, SLC5A9,TACSTD2, TAS1R2, TIE1, TLR5, TMEM81, TNFRSF14, TNFRSF1B, TRABD2B, USH2A,VCAM1, and ZP4.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 1. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ABCA4,ADAM30, AQP10, ASTN1, C1orf101, CACNA1S, CATSPER4, CD101, CD164L2, CD1A,CD1C, CD244, CD34, CD46, CELSR2, CHRNB2, CLCA2, CLDN19, CLSTN1, CR1,CR2, CRB1, CSF3R, CSMD2, ECE1, ELTD1, EMC1, EPHA10, EPHA2, EPHA8, ERMAP,FCAMR, FCER1A, FCGR1B, FCGR2A, FCGR2B, FCGR3A, FCRL1, FCRL3, FCRL4,FCRL5, FCRL6, GJB4, GPA33, GPR157, GPR37L1, GPR88, HCRTR1, IGSF3, IGSF9,IL22RA1, IL23R, ITGA10, KIAA1324, KIAA2013, LDLRAD2, LEPR, LGR6, LRIG2,LRP8, LRRC52, LRRC8B, LRRN2, LY9, MIA3, MR1, MUC1, MXRA8, NCSTN, NFASC,NOTCH2, NPR1, NTRK1, OPN3, OR10J1, OR10J4, OR10K1, OR1OR2, OR10T2,OR10X1, OR11L1, OR14A16, OR14I1, OR14K1, OR2AK2, OR2C3, OR2G2, OR2G3,OR2L2, OR2M7, OR2T12, OR2T27, OR2T1, OR2T3, OR2T29, OR2T33, OR2T34,OR2T35, OR2T3, OR2T4, OR2T5, OR2T6, OR2T7, OR2T8, OR2W3, OR6F1, OR6K2,OR6K3, OR6K6, OR6N1, OR6P1, OR6Y1, PDPN, PEAR1, PIGR, PLXNA2, PTCH2,PTCHD2, PTGFRN, PTPRC, PTPRF, PTGFRN, PVRL4, RHBG, RXFP4, S1PR1, SCNN1D,SDC3, SELE, SELL, SELP, SEMA4A, SEMA6C, SLAMF7, SLAMF9, SLC2A7, SLC5A9,TACSTD2, TAS1R2, TIE1, TLR5, TMEM81, TNFRSF14, TNFRSF1B, TRABD2B, USH2A,VCAM1, and ZP4.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 2. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ABCG5, ALK,ASPRV1, ATRAID, CD207, CD8B, CHRNG, CLEC4F, CNTNAP5, CRIM1, CXCR1, DNER,DPP10, EDAR, EPCAM, GPR113, GPR148, GPR35, GPR39, GYPC, IL1RL1, ITGA4,ITGA6, ITGAV, LCT, LHCGR, LRP1B, LRP2, LY75, MARCO, MERTK, NRP2, OR6B2,PLA2R1, PLB1, PROKR1, PROM2, SCN7A, SDC1, SLC23A3, SLC5A6, TGOLN2,THSD7B, TM4SF20, TMEFF2, TMEM178A, TPO, and TRABD2A.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 2. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ABCG5,ALK, ASPRV1, ATRAID, CD207, CD8B, CHRNG, CLEC4F, CNTNAP5, CRIM1, CXCR1,DNER, DPP10, EDAR, EPCAM, GPR113, GPR148, GPR35, GPR39, GYPC, IL1RL1,ITGA4, ITGA6, ITGAV, LCT, LHCGR, LRP1B, LRP2, LY75, MARCO, MERTK, NRP2,OR6B2, PLA2R1, PLB1, PROKR1, PROM2, SCN7A, SDC1, SLC23A3, SLC5A6,TGOLN2, THSD7B, TM4SF20, TMEFF2, TMEM178A, TPO, and TRABD2A.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 3. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ACKR2, ALCAM,ANO10, ATP13A4, BTLA, CACNA1D, CACNA2D2, CACNA2D3, CASR, CCRL2, CD200,CD200R1, CD86, CD96, CDCP1, CDHR4, CELSR3, CHL1, CLDN11, CLDN18, CLSTN2,CSPG5, CX3CR1, CXCR6, CYP8B1, DCBLD2, DRD3, EPHA6, EPHB3, GABRR3, GP5,GPR128, GPR15, GPR27, GRM2, GRM7, HEG1, HTR3C, HTR3D, HTR3E, IGSF11,IL17RC, IL17RD, IL17RE, IL5RA, IMPG2, ITGA9, ITGB5, KCNMB3, LRIG1,LRRC15, LRRN1, MST1R, NAALADL2, NRROS, OR5AC1, OR5H1, OR5H14, OR5H15,OR5H6, OR5K2, OR5K3, OR5K4, PIGX, PLXNB1, PLXND1, PRRT3, PTPRG, ROBO2,RYK, SEMA5B, SIDT1, SLC22A14, SLC33A1, SLC4A7, SLITRK3, STAB1, SUSD5,TFRC, TLR9, TMEM108, TMEM44, TMPRSS7, TNFSF10, UPK1B, VIPR1, and ZPLD1.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 3. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ACKR2,ALCAM, ANO10, ATP13A4, BTLA, CACNA1D, CACNA2D2, CACNA2D3, CASR, CCRL2,CD200, CD200R1, CD86, CD96, CDCP1, CDHR4, CELSR3, CHL1, CLDN11, CLDN18,CLSTN2, CSPG5, CX3CR1, CXCR6, CYP8B1, DCBLD2, DRD3, EPHA6, EPHB3,GABRR3, GP5, GPR128, GPR15, GPR27, GRM2, GRM7, HEG1, HTR3C, HTR3D,HTR3E, IGSF11, IL17RC, IL17RD, IL17RE, IL5RA, IMPG2, ITGA9, ITGB5,KCNMB3, LRIG1, LRRC15, LRRN1, MST1R, NAALADL2, NRROS, OR5AC1, OR5H1,OR5H14, OR5H15, OR5H6, OR5K2, OR5K3, OR5K4, PIGX, PLXNB1, PLXND1, PRRT3,PTPRG, ROBO2, RYK, SEMA5B, SIDT1, SLC22A14, SLC33A1, SLC4A7, SLITRK3,STAB1, SUSD5, TFRC, TLR9, TMEM108, TMEM44, TMPRSS7, TNFSF10, UPK1B,VIPR1, and ZPLD1.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 4. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ANTXR2, BTC,CNGA1, CORIN, EGF, EMCN, ENPEP, EPHA5, ERVMER34-1, EVC2, FAT1, FAT4,FGFRL1, FRAS1, GPR125, GRID2, GYPA, GYPB, KDR, KIAA0922, KLB, MFSD8,PARM1, PDGFRA, RNF150, TENM3, TLR10, TLR1, TLR6, TMEM156, TMPRSS11A,TMPRSS11B, TMPRSS11E, TMPRSS11F, UGT2A1, and UNC5C.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 4. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ANTXR2,BTC, CNGA1, CORIN, EGF, EMCN, ENPEP, EPHA5, ERVMER34-1, EVC2, FAT1,FAT4, FGFRL1, FRAS1, GPR125, GRID2, GYPA, GYPB, KDR, KIAA0922, KLB,MFSD8, PARM1, PDGFRA, RNF150, TENM3, TLR10, TLR1, TLR6, TMEM156,TMPRSS11A, TMPRSS11B, TMPRSS11E, TMPRSS11F, UGT2A1, and UNC5C.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 5. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ADAM19, ADRB2,BTNL3, BTNL8, BTNL9, C5orf15, CATSPER3, CD180, CDH12, CDHR2, COL23A1,CSF1R, F2RL2, FAM174A, FAT2, FGFR4, FLT4, GABRA6, GABRG2, GPR151, GPR98,GRM6, HAVCR1, HAVCR2, IL31RA, IL6ST, IL7R, IQGAP2, ITGA1, ITGA2, KCNMB1,LIFR, LNPEP, MEGF10, NIPAL4, NPR3, NRG2, OR2V1, OR2Y1, OSMR, PCDH12,PCDH1, PCDHA1, PCDHA2, PCDHA4, PCDHA8, PCDHA9, PCDHB10, PCDHB11,PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB2, PCDHB3, PCDHB4, PCDHB5,PCDHB6, PCDHGA1, PCDHGA4, PDGFRB, PRLR, SEMA5A, SEMA6A, SGCD, SLC1A3,SLC22A4, SLC22A5, SLC23A1, SLC36A3, SLC45A2, SLC6A18, SLC6A19, SLCO6A1,SV2C, TENM2, TIMD4, and UGT3A1.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 5. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ADAM19,ADRB2, BTNL3, BTNL8, BTNL9, C5orf15, CATSPER3, CD180, CDH12, CDHR2,COL23A1, CSF1R, F2RL2, FAM174A, FAT2, FGFR4, FLT4, GABRA6, GABRG2,GPR151, GPR98, GRM6, HAVCR1, HAVCR2, IL31RA, IL6ST, IL7R, IQGAP2, ITGA1,ITGA2, KCNMB1, LIFR, LNPEP, MEGF10, NIPAL4, NPR3, NRG2, OR2V1, OR2Y1,OSMR, PCDH12, PCDH1, PCDHA1, PCDHA2, PCDHA4, PCDHA8, PCDHA9, PCDHB10,PCDHB11, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB2, PCDHB3, PCDHB4,PCDHB5, PCDHB6, PCDHGA1, PCDHGA4, PDGFRB, PRLR, SEMA5A, SEMA6A, SGCD,SLC1A3, SLC22A4, SLC22A5, SLC23A1, SLC36A3, SLC45A2, SLC6A18, SLC6A19,SLCO6A1, SV2C, TENM2, TIMD4, and UGT3A1.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 6. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of BAI3, BTN1A1,BTN2A1, BTN2A2, BTN3A1, BTN3A2, BTNL2, CD83, DCBLD1, DLL1, DPCR1, ENPP1,ENPP3, ENPP4, EPHA7, GABBR1, GABRR1, GCNT6, GFRAL, GJB7, GLP1R, GPR110,GPR111, GPR116, GPR126, GPR63, GPRC6A, HFE, HLA-A, HLA-B, HLA-C,HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2,HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-G, IL20RA, ITPR3, KIAA0319,LMBRD1, LRFN2, LRP11, MAS1L, MEP1A, MICA, MICB, MOG, MUC21, MUC22, NCR2,NOTCH4, OPRM1, OR10C1, OR12D2, OR12D3, OR14J1, OR2B2, OR2B6, OR2J1,OR2W1, OR5V1, PDE10A, PI16, PKHD1, PTCRA, PTK7, RAET1E, RAET1G, ROS1,SDIM1, SLC16A10, SLC22A1, SLC44A4, TAAR2, TREM1, TREML1, and TREML2.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 6. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of BAI3,BTN1A1, BTN2A1, BTN2A2, BTN3A1, BTN3A2, BTNL2, CD83, DCBLD1, DLL1,DPCR1, ENPP1, ENPP3, ENPP4, EPHA7, GABBR1, GABRR1, GCNT6, GFRAL, GJB7,GLP1R, GPR110, GPR111, GPR116, GPR126, GPR63, GPRC6A, HFE, HLA-A, HLA-B,HLA-C, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1,HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-G, IL20RA, ITPR3,KIAA0319, LMBRD1, LRFN2, LRP11, MAS1L, MEP1A, MICA, MICB, MOG, MUC21,MUC22, NCR2, NOTCH4, OPRM1, OR10C1, OR12D2, OR12D3, OR14J1, OR2B2,OR2B6, OR2J1, OR2W1, OR5V1, PDE10A, PI16, PKHD1, PTCRA, PTK7, RAET1E,RAET1G, ROS1, SDIM1, SLC16A10, SLC22A1, SLC44A4, TAAR2, TREM1, TREML1,and TREML2.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 7. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of AQP1, C7orf50,CD36, CDHR3, CNTNAP2, DPP6, EGFR, EPHA1, EPHB6, ERVW-1, GHRHR, GJC3,GPNMB, GRM8, HUS1, HYAL4, KIAA1324L, LRRN3, MET, MUC12, MUC17, NPC1L1,NPSR1, OR2A12, OR2A14, OR2A25, OR2A42, OR2A7, OR2A2, OR2AE1, OR2F2,OR6V1, PILRA, PILRB, PKD1L1, PLXNA4, PODXL, PTPRN2, PTPRZ1, RAMP3,SLC29A4, SMO, TAS2R16, TAS2R40, TAS2R4, TFR2, THSD7A, TMEM213, TTYH3,ZAN, and ZP3.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 7. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of AQP1,C7orf50, CD36, CDHR3, CNTNAP2, DPP6, EGFR, EPHA1, EPHB6, ERVW-1, GHRHR,GJC3, GPNMB, GRM8, HUS1, HYAL4, KIAA1324L, LRRN3, MET, MUC12, MUC17,NPC1L1, NPSR1, OR2A12, OR2A14, OR2A25, OR2A42, OR2A7, OR2A2, OR2AE1,OR2F2, OR6V1, PILRA, PILRB, PKD1L1, PLXNA4, PODXL, PTPRN2, PTPRZ1,RAMP3, SLC29A4, SMO, TAS2R16, TAS2R40, TAS2R4, TFR2, THSD7A, TMEM213,TTYH3, ZAN, and ZP3.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 8. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ADAM18,ADAM28, ADAM32, ADAM7, ADAM9, ADRA1A, CDH17, CHRNA2, CSMD1, CSMD3,DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1, PRSS55, SCARA3, SCARA5,SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A1, TNFRSF10A, and TNFRSF10B.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 8. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ADAM18,ADAM28, ADAM32, ADAM7, ADAM9, ADRA1A, CDH17, CHRNA2, CSMD1, CSMD3,DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1, PRSS55, SCARA3, SCARA5,SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A1, TNFRSF10A, and TNFRSF10B.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 9. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ABCA1, AQP7,ASTN2, C9orf135, CA9, CD72, CNTNAP3B, CNTNAP3, CRB2, ENTPD8, GPR144,GRIN3A, IZUMO3, KIAA1161, MAMDC4, MEGF9, MUSK, NOTCH1, OR13C2, OR13C3,OR13C5, OR13C8, OR13C9, OR13D1, OR13F1, OR1B1, OR1J2, OR1K1, OR1L1,OR1L3, OR1L6, OR1L8, OR1N1, OR1N2, OR1Q1, OR2S2, PCSK5, PDCD1LG2,PLGRKT, PTPRD, ROR2, SEMA4D, SLC31A1, TEK, TLR4, TMEM2, and VLDLR.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 9. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ABCA1,AQP7, ASTN2, C9orf135, CA9, CD72, CNTNAP3B, CNTNAP3, CRB2, ENTPD8,GPR144, GRIN3A, IZUMO3, KIAA1161, MAMDC4, MEGF9, MUSK, NOTCH1, OR13C2,OR13C3, OR13C5, OR13C8, OR13C9, OR13D1, OR13F1, OR1B1, OR1J2, OR1K1,OR1L1, OR1L3, OR1L6, OR1L8, OR1N1, OR1N2, OR1Q1, OR2S2, PCSK5, PDCD1LG2,PLGRKT, PTPRD, ROR2, SEMA4D, SLC31A1, TEK, TLR4, TMEM2, and VLDLR.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 10. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ABCC2, ADAM8,ADRB1, ANTXRL, ATRNL1, C10orf54, CDH23, CDHR1, CNNM2, COL13A1, COL17A1,ENTPD1, FZD8, FGFR2, GPR158, GRID1, IL15RA, IL2RA, ITGA8, ITGB1, MRC1,NRG3, NPFFR1, NRP1, OPN4, PCDH15, PKD2L1, PLXDC2, PRLHR, RET, RGR,SLC16A9, SLC29A3, SLC39A12, TACR2, TCTN3, TSPAN15, UNC5B, and VSTM4.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 10. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ABCC2,ADAM8, ADRB1, ANTXRL, ATRNL1, C10orf54, CDH23, CDHR1, CNNM2, COL13A1,COL17A1, ENTPD1, FZD8, FGFR2, GPR158, GRID1, IL15RA, IL2RA, ITGA8,ITGB1, MRC1, NRG3, NPFFR1, NRP1, OPN4, PCDH15, PKD2L1, PLXDC2, PRLHR,RET, RGR, SLC16A9, SLC29A3, SLC39A12, TACR2, TCTN3, TSPAN15, UNC5B, andVSTM4.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 11. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of AMICA1, ANO1,ANO3, APLP2, C11orf24, CCKBR, CD248, CD44, CD5, CD6, CD82, CDON, CLMP,CRTAM, DCHS1, DSCAML1, FAT3, FOLH1, GDPD4, GDPD5, GRIK4, HEPHL1, HTR3B,IFITM10, IL10RA, KIRREL3, LGR4, LRP4, LRP5, LRRC32, MCAM, MFRP, MMP26,MPEG1, MRGPRE, MRGPRF, MRGPRX2, MRGPRX3, MRGPRX4, MS4A4A, MS4A6A,MTNR1B, MUC15, NAALAD2, NAALADL1, NCAM1, NRXN2, OR10A2, OR10A5, OR10A6,OR10D3, OR10G4, OR10G7, OR10G8, OR10G9, OR10Q1, OR10S1, OR1S1, OR2AG1,OR2AG2, OR2D2, OR4A47, OR4A15, OR4A5, OR4C11, OR4C13, OR4C15, OR4C16,OR4C3, OR4C46, OR4C5, OR4D6, OR4A8P, OR4D9, OR4S2, OR4X1, OR51E1,OR51L1, OR52A1, OR52E1, OR52E2, OR52E4, OR52E6, OR52I1, OR52I2, OR52J3,OR52L1, OR52N1, OR52N2, OR52N4, OR52W1, OR56B1, OR56B4, OR5A1, OR5A2,OR5AK2, OR5AR1, OR5B17, OR5B3, OR5D14, OR5D16, OR5D18, OR5F1, OR5I1,OR5L2, OR5M11, OR5M3, OR5P2, OR5R1, OR5T2, OR5T3, OR5W2, OR6A2, OR6T1,OR6X1, OR8A1, OR8B12, OR8B2, OR8B3, OR8B4, OR8D1, OR8D2, OR8H1, OR8H2,OR8H3, OR8I2, OR8J1, OR8J2, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR9G1,OR9G4, OR9Q2, P2RX3, PTPRJ, ROBO3, SIGIRR, SLC22A10, SLC3A2, SLC5A12,SLCO2B1, SORL1, ST14, SYT8, TENM4, TMEM123, TMEM225, TMPRSS4, TMPRSS5,TRIM5, TRPM5, TSPAN18, and ZP1.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 11. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of AMICA1,ANO1, ANO3, APLP2, C11orf24, CCKBR, CD248, CD44, CD5, CD6, CD82, CDON,CLMP, CRTAM, DCHS1, DSCAML1, FAT3, FOLH1, GDPD4, GDPD5, GRIK4, HEPHL1,HTR3B, IFITM10, IL10RA, KIRREL3, LGR4, LRP4, LRP5, LRRC32, MCAM, MFRP,MMP26, MPEG1, MRGPRE, MRGPRF, MRGPRX2, MRGPRX3, MRGPRX4, MS4A4A, MS4A6A,MTNR1B, MUC15, NAALAD2, NAALADL1, NCAM1, NRXN2, OR10A2, OR10A5, OR10A6,OR10D3, OR10G4, OR10G7, OR10G8, OR10G9, OR10Q1, OR10S1, OR1S1, OR2AG1,OR2AG2, OR2D2, OR4A47, OR4A15, OR4A5, OR4C11, OR4C13, OR4C15, OR4C16,OR4C3, OR4C46, OR4C5, OR4D6, OR4A8P, OR4D9, OR4S2, OR4X1, OR51E1,OR51L1, OR52A1, OR52E1, OR52E2, OR52E4, OR52E6, OR52I1, OR52I2, OR52J3,OR52L1, OR52N1, OR52N2, OR52N4, OR52W1, OR56B1, OR56B4, OR5A1, OR5A2,OR5AK2, OR5AR1, OR5B17, OR5B3, OR5D14, OR5D16, OR5D18, OR5F1, OR5I1,OR5L2, OR5M11, OR5M3, OR5P2, OR5R1, OR5T2, OR5T3, OR5W2, OR6A2, OR6T1,OR6X1, OR8A1, OR8B12, OR8B2, OR8B3, OR8B4, OR8D1, OR8D2, OR8H1, OR8H2,OR8H3, OR8I2, OR8J1, OR8J2, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR9G1,OR9G4, OR9Q2, P2RX3, PTPRJ, ROBO3, SIGIRR, SLC22A10, SLC3A2, SLC5A12,SLCO2B1, SORL1, ST14, SYT8, TENM4, TMEM123, TMEM225, TMPRSS4, TMPRSS5,TRIM5, TRPM5, TSPAN18, and ZP1.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 12. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ANO4, AVPR1A,BCL2L14, CACNA2D4, CD163, CD163L1, CD27, CD4, CLEC12A, CLEC1B, CLEC2A,CLEC4C, CLEC7A, CLECL1, CLSTN3, GPR133, GPRC5D, ITGA7, ITGB7, KLRB1,KLRC2, KLRC3, KLRC4, KLRF1, KLRF2, LRP1, LRP6, MANSC1, MANSC4, OLR1,OR10AD1, OR10P1, OR2AP1, OR6C1, OR6C2, OR6C3, OR6C4, OR6C6, OR6C74,OR6C76, OR8S1, OR9K2, ORAI1, P2RX4, P2RX7, PRR4, PTPRB, PTPRQ, PTPRR,SCNN1A, SELPLG, SLC2A14, SLC38A4, SLC5A8, SLC6A15, SLC8B1, SLCO1A2,SLCO1B1, SLCO1B7, SLCO1C1, SSPN, STAB2, TAS2R10, TAS2R13, TAS2R14,TAS2R20, TAS2R30, TAS2R31, TAS2R42, TAS2R43, TAS2R46, TAS2R7, TMEM119,TMEM132B, TMEM132C, TMEM132D, TMPRSS12, TNFRSF1A, TSPAN8, and VSIG10.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 12. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ANO4,AVPR1A, BCL2L14, CACNA2D4, CD163, CD163L1, CD27, CD4, CLEC12A, CLEC1B,CLEC2A, CLEC4C, CLEC7A, CLECL1, CLSTN3, GPR133, GPRC5D, ITGA7, ITGB7,KLRB1, KLRC2, KLRC3, KLRC4, KLRF1, KLRF2, LRP1, LRP6, MANSC1, MANSC4,OLR1, OR10AD1, OR10P1, OR2AP1, OR6C1, OR6C2, OR6C3, OR6C4, OR6C6,OR6C74, OR6C76, OR8S1, OR9K2, ORAI1, P2RX4, P2RX7, PRR4, PTPRB, PTPRQ,PTPRR, SCNN1A, SELPLG, SLC2A14, SLC38A4, SLC5A8, SLC6A15, SLC8B1,SLCO1A2, SLCO1B1, SLCO1B7, SLCO1C1, SSPN, STAB2, TAS2R10, TAS2R13,TAS2R14, TAS2R20, TAS2R30, TAS2R31, TAS2R42, TAS2R43, TAS2R46, TAS2R7,TMEM119, TMEM132B, TMEM132C, TMEM132D, TMPRSS12, TNFRSF1A, TSPAN8, andVSIG10.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 13. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ATP4B, ATP7B,FLT3, FREM2, HTR2A, KL, PCDH8, RXFP2, SGCG, SHISA2, SLC15A1, SLITRK6,and TNFRSF19.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 13. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ATP4B,ATP7B, FLT3, FREM2, HTR2A, KL, PCDH8, RXFP2, SGCG, SHISA2, SLC15A1,SLITRK6, and TNFRSF19.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 14. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ADAM21,BDKRB2, C14orf37, CLEC14A, DLK1, FLRT2, GPR135, GPR137C, JAG2, LTB4R2,MMP14, OR11G2, OR11H12, OR11H6, OR4K1, OR4K15, OR4K5, OR4L1, OR4N2,OR4N5, SLC24A4, and SYNDIG1L.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 14. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ADAM21,BDKRB2, C14orf37, CLEC14A, DLK1, FLRT2, GPR135, GPR137C, JAG2, LTB4R2,MMP14, OR11G2, OR11H12, OR11H6, OR4K1, OR4K15, OR4K5, OR4L1, OR4N2,OR4N5, SLC24A4, and SYNDIG1L.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 15. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ANPEP, CD276,CHRNA7, CHRNB4, CSPG4, DUOX1, DUOX2, FAM174B, GLDN, IGDCC4, ITGA11,LCTL, LTK, LYSMD4, MEGF11, NOX5, NRG4, OCA2, OR4F4, OR4M2, OR4N4, PRTG,RHCG, SCAMPS, SEMA4B, SEMA6D, SLC24A1, SLC24A5, SLC28A1, SPG11, STRA6,TRPM1, and TYRO3.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 15. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ANPEP,CD276, CHRNA7, CHRNB4, CSPG4, DUOX1, DUOX2, FAM174B, GLDN, IGDCC4,ITGA11, LCTL, LTK, LYSMD4, MEGF11, NOX5, NRG4, OCA2, OR4F4, OR4M2,OR4N4, PRTG, RHCG, SCAMPS, SEMA4B, SEMA6D, SLC24A1, SLC24A5, SLC28A1,SPG11, STRA6, TRPM1, and TYRO3.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 16. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ATP2C2,CACNA1H, CD19, CDH11, CDH15, CDH16, CDH3, CDH5, CNGB1, CNTNAP4, GDPD3,GPR56, GPR97, IFT140, IL4R, ITFG3, ITGAL, ITGAM, ITGAX, KCNG4, MMP15,MSLNL, NOMO1, NOMO3, OR2C1, PIEZO1, PKD1, PKD1L2, QPRT, SCNN1B, SEZ6L2,SLC22A31, SLC5A11, SLC7A6, SPN, TMC5, TMC7, TMEM204, TMEM219, andTMEM8A.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 16. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ATP2C2,CACNA1H, CD19, CDH11, CDH15, CDH16, CDH3, CDH5, CNGB1, CNTNAP4, GDPD3,GPR56, GPR97, IFT140, IL4R, ITFG3, ITGAL, ITGAM, ITGAX, KCNG4, MMP15,MSLNL, NOMO1, NOMO3, OR2C1, PIEZO1, PKD1, PKD1L2, QPRT, SCNN1B, SEZ6L2,SLC22A31, SLC5A11, SLC7A6, SPN, TMC5, TMC7, TMEM204, TMEM219, andTMEM8A.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 17. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ABCC3, ACE,AOC3, ARL17B, ASGR2, C17orf80, CD300A, CD300C, CD300E, CD300LF, CD300LG,CHRNB1, CLEC10A, CNTNAP1, CPD, CXCL16, ERBB2, FAM171A2, GCGR, GLP2R,GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3, ITGAE, ITGB3, KCNJ12, LRRC37A2,LRRC37A3, LRRC37A, LRRC37B, MRC2, NGFR, OR1A2, OR1D2, OR1G1, OR3A1,OR3A2, OR4D1, OR4D2, RNF43, SCARF1, SCN4A, SDK2, SECTM1, SEZ6, SHPK,SLC26A11, SLC5A10, SPACA3, TMEM102, TMEM132E, TNFSF12, TRPV3, TTYH2, andTUSC5.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 17. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ABCC3,ACE, AOC3, ARL17B, ASGR2, C17orf80, CD300A, CD300C, CD300E, CD300LF,CD300LG, CHRNB1, CLEC10A, CNTNAP1, CPD, CXCL16, ERBB2, FAM171A2, GCGR,GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3, ITGAE, ITGB3, KCNJ12,LRRC37A2, LRRC37A3, LRRC37A, LRRC37B, MRC2, NGFR, OR1A2, OR1D2, OR1G1,OR3A1, OR3A2, OR4D1, OR4D2, RNF43, SCARF1, SCN4A, SDK2, SECTM1, SEZ6,SHPK, SLC26A11, SLC5A10, SPACA3, TMEM102, TMEM132E, TNFSF12, TRPV3,TTYH2, and TUSC5.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 18. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of APCDD1, CDH19,CDH20, CDH7, COLEC12, DCC, DSC1, DSG1, DSG3, DYNAP, MEP1B, PTPRM,SIGLEC15, and TNFRSF11A.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 18. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of APCDD1,CDH19, CDH20, CDH7, COLEC12, DCC, DSC1, DSG1, DSG3, DYNAP, MEP1B, PTPRM,SIGLEC15, and TNFRSF11A.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 19. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ABCA7, ACPT,BCAM, C19orf38, C19orf59, C5AR1, CATSPERD, CATSPERG, CD22, CD320, CD33,CD97, CEACAM19, CEACAM1, CEACAM21, CEACAM3, CEACAM4, CLEC4M, DLL3, EMR1,EMR2, EMR3, ERVV-1, ERVV-2, FAM187B, FCAR, FFAR3, FPR1, FXYD5, GFY, GP6,GPR42, GRIN3B, ICAM3, IGFLR1, IL12RB1, IL27RA, KIR2DL1, KIR2DL3,KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KIRREL2, KISS1R, LAIR1, LDLR,LILRA1, LILRA2, LILRA4, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5,LINGO3, LPHN1, LRP3, MADCAM1, MAG, MEGF8, MUC16, NCR1, NOTCH3, NPHS1,OR10H1, OR10H2, OR10H3, OR10H4, OR1I1, OR2Z1, OR7A10, OR7C1, OR7D4,OR7E24, OR7G1, OR7G2, OR7G3, PLVAP, PTGIR, PTPRH, PTPRS, PVR, SCN1B,SHISA7, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC5, SIGLEC6, SIGLEC8,SIGLEC9, SLC44A2, SLC5A5, SLC7A9, SPINT2, TARM1, TGFBR3L, TMC4, TMEM91,TMEM161A, TMPRSS9, TNFSF14, TNFSF9, TRPM4, VN1R2, VSIG10L, VSTM2B, andZNRF4.

In some embodiments, the the recognition moiety for use in the the iCARor pCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 19. Insome embodiments, the recognition moiety for use in the the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ABCA7,ACPT, BCAM, C19orf38, C19orf59, C5AR1, CATSPERD, CATSPERG, CD22, CD320,CD33, CD97, CEACAM19, CEACAM1, CEACAM21, CEACAM3, CEACAM4, CLEC4M, DLL3,EMR1, EMR2, EMR3, ERVV-1, ERVV-2, FAM187B, FCAR, FFAR3, FPR1, FXYD5,GFY, GP6, GPR42, GRIN3B, ICAM3, IGFLR1, IL12RB1, IL27RA, KIR2DL1,KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KIRREL2, KISS1R, LAIR1,LDLR, LILRA1, LILRA2, LILRA4, LILRA6, LILRB1, LILRB2, LILRB3, LILRB4,LILRB5, LINGO3, LPHN1, LRP3, MADCAM1, MAG, MEGF8, MUC16, NCR1, NOTCH3,NPHS1, OR10H1, OR10H2, OR10H3, OR10H4, OR1I1, OR2Z1, OR7A10, OR7C1,OR7D4, OR7E24, OR7G1, OR7G2, OR7G3, PLVAP, PTGIR, PTPRH, PTPRS, PVR,SCN1B, SHISA7, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC5, SIGLEC6, SIGLEC8,SIGLEC9, SLC44A2, SLC5A5, SLC7A9, SPINT2, TARM1, TGFBR3L, TMC4, TMEM91,TMEM161A, TMPRSS9, TNFSF14, TNFSF9, TRPM4, VN1R2, VSIG10L, VSTM2B, andZNRF4.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 20. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ABHD12,ADAM33, ADRA1D, APMAP, ATRN, CD40, CD93, CDH22, CDH26, CDH4, FLRT3,GCNT7, GGT7, JAG1, LRRN4, NPBWR2, OCSTAMP, PTPRA, PTPRT, SEL1L2,SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3, SLC2A10, SLC4A11, SSTR4, andTHBD.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 20. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of ABHD12,ADAM33, ADRA1D, APMAP, ATRN, CD40, CD93, CDH22, CDH26, CDH4, FLRT3,GCNT7, GGT7, JAG1, LRRN4, NPBWR2, OCSTAMP, PTPRA, PTPRT, SEL1L2,SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3, SLC2A10, SLC4A11, SSTR4, andTHBD.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 21. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of CLDN8, DSCAM,ICOSLG, IFNAR1, IFNGR2, IGSF5, ITGB2, KCNJ15, NCAM2, SLC19A1, TMPRSS15,TMPRSS2, TMPRSS3, TRPM2, and UMODL1.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 21. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting of CLDN8,DSCAM, ICOSLG, IFNAR1, IFNGR2, IGSF5, ITGB2, KCNJ15, NCAM2, SLC19A1,TMPRSS15, TMPRSS2, TMPRSS3, TRPM2, and UMODL1.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome 22. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of CACNA1I,CELSR1, COMT, CSF2RB, GGT1, GGT5, IL2RB, KREMEN1, MCHR1, OR11H1, P2RX6,PKDREJ, PLXNB2, SCARF2, SEZ6L, SSTR3, SUSD2, TMPRSS6, and TNFRSF13C.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome 22. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting ofCACNA1I, CELSR1, COMT, CSF2RB, GGT1, GGT5, IL2RB, KREMEN1, MCHR1,OR11H1, P2RX6, PKDREJ, PLXNB2, SCARF2, SEZ6L, SSTR3, SUSD2, TMPRSS6, andTNFRSF13C.

In some embodiments, the the recognition moiety for use in the aCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from chromosome X. In someembodiments, the recognition moiety for use in the aCAR providesspecifity to at least one extracellular polymorphic epitope in a geneproduct from a gene selected from the group consisting of ATP6AP2,ATP7A, CNGA2, EDA2R, FMR1NB, GLRA4, GPR112, GUCY2F, HEPH, P2RY10, P2RY4,PLXNA3, PLXNB3, TLR8, VSIG4, and XG.

In some embodiments, the the recognition moiety for use in the iCAR orpCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from chromosome X. Insome embodiments, the recognition moiety for use in the iCAR or pCARprovides specifity to at least one extracellular polymorphic epitope ina gene product from a gene selected from the group consisting ofATP6AP2, ATP7A, CNGA2, EDA2R, FMR1NB, GLRA4, GPR112, GUCY2F, HEPH,P2RY10, P2RY4, PLXNA3, PLXNB3, TLR8, VSIG4, and XG.

The sequences encoding the variable regions of these antibodies caneasily be cloned from the relevant hybridoma and used for constructinggenes encoding scFvs against any desired target, including for example,scFvs against specific HLA Class-I allelic epitope variants, and whichwould be suitable for incorporation into a CAR construct using toolswidely available as disclosed e.g., in Molecular Cloning: A LaboratoryManual (Fourth Edition) Green and Sambrook, Cold Spring HarborLaboratory Press; Antibodies: A Laboratory Manual (Second Edition),Edited by Edward A. Greenfield, 2012 CSH laboratory press; UsingAntibodies, A laboratory manual by Ed Harlow and David Lane, 1999 CSHlaboratory press.

The present invention provides a database comprising DNA sequences ofpolymorphic variants lost in tumor cells due to LOH, and that encodecell-surface products, wherein the variation at the DNA sequence resultsin a variation at the amino acid sequence in an extracellular domain ofthe encoded protein. The information was retrieved from severaldatabases open to the general public, such as TCGA, available on thepublic National Institute of Health TCGA data portal(https://gdc.cancer.gov/), which provides, inter alia, data that can beused to infer relative copy number of the gene in a variety of tumortypes and the cbio portal for TCGA data at http://www.cbioportal.org(Cerami et al., 2012, Gao et al., 2013); the Exome AggregationConsortium (ExAC) database (exac.broadinstitute.org, Lek et al., 2016),providing, inter alia, allele frequencies of SNP variants in variouspopulations; the Genotype-Tissue Expression (GTEX) database v6p (dbGaPAccession phs000424.v6.p1) (https://gtexportal.org/home, ConsortiumGT._Human genomics, 2015) which includes tissue expression data forgenes; and databases providing structural information of proteins, suchas the Human Protein Atlas (Uhlen et al., 2015); the Cell SurfaceProtein Atlas (Bausch-Fluck et al., 2015), a mass-spectrometry baseddatabase of N-glycosylated cell-surface proteins, and the UniProtdatabase (www.uniprot.org/downloads).

The present invention further provides a method for genome-wideidentification of genes that encode expressed cell-surface proteins thatundergo LOH. The identified genes must meet the following criteria: 1)The gene encodes a transmembrane protein—therefore having a portionexpressed on the cell surface to allow the iCAR or pCAR binding; 2) Thegene has at least two expressed alleles (in at least one ethnicpopulation checked); 3) The allelic variation found for that gene causesan amino acid change relative to the reference sequence in anextracellular region of the protein; 4) The gene is located in achromosomal region which undergoes LOH in cancer; 5) The gene isexpressed in a tissue-of-origin of a tumor type in which thecorresponding region was found to undergo LOH.

In principle genes as described above, suitable to encode targets foriCAR or pCAR binding may be identified by any method known in the art,and not only by database mining. For example, the concept of LOH is notnew and LOH information for specific genes, chromosomes, orgenomic/chromosomal regions in specific tumors has already beenpublished in the literature and candidate genes can therefore be derivedfrom the available publications. Alternatively, such information can befound by whole genome hybridizations with chromosomal markers such asmicrosatellite probes (Medintz et al., 2000, Genome Res. 2000 August;10(8): 1211-1218) or by any other suitable method (Ramos and Amorim,2015, J. Bras. Patol. Med. Lab. 51(3):198-196).

Similarly, information regarding allelic variants is publicly availablein various databases, and can also be easily obtained for a personalizedcase by genomic sequencing of a suspected region. Also, informationregarding protein structure and expression pattern is publicly availableand easily accessible as described above.

Accordingly, as information regarding the various criteria for manygenes and SNPs is publicly available and the techniques for retrievingit are generally known, the main novelty of the application is using LOHas a criterion for choosing a target for iCAR or pCAR recognition, andthe concept of personalizing treatment based on a specific allele lostin a specific patient.

As a non-limiting example, it was found according to the presentinvention that HLA genes, including non-classical HLA-I and HLA-II genes(e.g., HLA-A, HLA-B HLA-C, HLA-E, HLA-F, HLA-G, HLA-DM, HLA-DO, HLA-DP,HLA-DQ, HLA-DR HLA-K and/or HLA-L) LOH, at varying frequencies, is arelatively frequent event in many tumor types (see FIGS. 10A-C), whichwould make these genes good candidates to be used as targets foriCAR/pCAR recognition for the purpose of the present invention.

The recognition of the aCAR target on normal cells in any healthyessential tissue in the absence of the pCAR or iCAR target would bedetrimental and is strictly forbidden. In this respect, the concept ofpCAR-aCAR or iCAR-aCAR pairs, as proposed here, constitutes a fail-safeactivation switch, as: i) cells not expressing the selected gene (incase the aCAR and the pCAR or iCAR target different products of the samegene) will not be targeted due to absence of the aCAR target antigen;ii) normal cells expressing this same gene will co-express both allelesand will not be targeted owing to the dominance of the pCAR or iCAR;iii) in case the pCAR or iCAR targets the product of a polymorphichousekeeping gene, all cells in the body will be protected; and iv) onlytumor cells which express the aCAR target but not the pCAR or iCAR onewill be attacked. In some embodiments, the recognition of the aCARtarget on normal cells in any healthy essential tissue in the absence ofthe pCAR or iCAR target would be detrimental. In some embodiments, cellsnot expressing the selected gene (in case the aCAR and the pCAR or iCARtarget different products of the same gene) will not be targeted due toabsence of the aCAR target antigen. In some embodiments, normal cellsexpressing this same gene will co-express both alleles and will not betargeted owing to the dominance of the pCAR or iCAR. In someembodiments, when the pCAR or iCAR targets the product of a polymorphichousekeeping gene, all cells in the body will be protected. In someembodiments, only tumor cells which express the aCAR target but not thepCAR or iCAR one will be attacked. In some embodiments, cells thatexpress both the aCAR/iCAR pair targets or both aCAR/pCAR pair targetswill be protected.

As emphasized above, according to the invention there must be permanentdominance of the inhibitory signal over the activating signal. It istherefore necessarily to ensure that no aCAR gene is expressed in agiven killer cell, at any time, in the absence of its iCAR partner. Thismay be implemented through the tandem assembly of these iCAR-aCAR genepairs as single-chain products or via a suitable bi-cistronic modalitybased, for example, on an internal ribosome entry site or on one ofseveral viral self-cleaving 2A peptides. As suggested by the vast bulkof data reported on bi-cistronic expression, the iCAR gene will alwaysbe positioned upstream of its aCAR partner to guarantee favorablestoichiometry. Another option would be engineering the killer cells toexpress both aCAR and iCAR or pCAR by transfecting or transducing thekiller cell with two independent constructs, each construct coding foreither aCAR or iCAR/pCAR. Of course, this is not an issue when using apCAR-aCAR gene pair. In some embodiments, the inhibitory signal isdominant over the activating signal. In some embodiments, the aCAR andiCAR or pCAR are expressed simultaneously in the same cell.

Another attractive option for assuring iCAR dominance is detaching theaCAR recognition moiety from its activating/costimulatory portion sothat both entities can only be assembled into one functional receptor inthe presence of a heterodimerizing small molecule. The ability totightly control the operative state of such split receptors by precisetiming, dosage and location was recently demonstrated in the context ofantitumor CARs (Wu et al., 2015).

In addition, the expected dominance is also likely to be intrinsic tothe particular composition of the iCAR signaling elements incorporatedinto the intracellular portion in the selected iCAR design that should‘compete’ with the signaling strength of the chosen aCAR platform. Thiscapacity will also be influenced by the relative affinities of the tworecognition moieties for their respective target epitopes (which wasdealt with above) and the overall avidities of their interactions.Concerning the latter, the proposed strategy secures both a favorableiCAR/aCAR stoichiometry and a balanced distribution of their respectivetarget epitopes on normal cells. Again, this is not an issue when usinga pCAR-aCAR gene pair.

To further assure safety, other conventional means currently implementedin the field of CAR and TCR immunotherapy can be employed, such as theuse of suicide genes or the use of mRNA electroporation for transientexpression.

While LOH often leaves the cells with only one allele of a given gene,it is frequently accompanied by duplication of the remaining chromosome,or chromosome part, resulting in ‘copy number neutral’-LOH (Lo et al.,2008; O'Keefe et al., 2010; Sathirapongsasuti et al., 2011). Under thesecircumstances, the emergence of epitope-loss variants requires twoindependent events and is thus less likely. Expressing several pCAR-aCARor iCAR-aCAR pairs in different fractions of the gene-modified cellswill prevent the appearance of mutational escapees even in ‘copy numberloss’ LOH cases, in which only a single copy of the target allele hasbeen retained. Yet, as single-copy genes may become essential, theirfunctional loss would be far less likely.

In view of the above, in one aspect, the present invention provides anucleic acid molecule comprising a nucleotide sequence encoding aninhibitory chimeric antigen receptor (iCAR) capable of preventing orattenuating undesired activation of an effector immune cell, wherein theiCAR comprises an extracellular domain that specifically binds to asingle allelic variant of a polymorphic cell surface epitope absent frommammalian tumor cells due to loss of heterozygosity (LOH) but present atleast on all cells of related mammalian normal tissue, or on vitalorgans the aCAR is expressed in; and an intracellular domain comprisingat least one signal transduction element that inhibits an effectorimmune cell.

In some embodiments, the polymorphic cell surface epitope is part of anantigen encoded by a tumor suppressor gene or a gene genetically linkedto a tumor suppressor gene, since such genes are likely to be lost dueto LOH in tumors. Additionally, the polymorphic cell surface epitope maybe part of an antigen encoded by a gene normally residing on achromosome or chromosomal arm that often undergo LOH in cancer cellssuch as, but not limited to, chromosomal arms 3p, 6p, 9p, 10q, 17p, 17q,or 18q, or chromosome 19. These epitopes can readily be identified inthe relevant databases as described herein.

In some embodiments, the polymorphic cell surface epitope is of ahousekeeping gene product, such as the unclassified AP2S1, CD81, GPAA1,LGALS9, MGAT2, MGAT4B, VAMP3; the cell adhesion proteins CTNNA1NM_001903, CTNNB1, CTNNBIP1 NM_020248, CTNNBL1 NM_030877, CTNND1NM_001085458 delta catenin; the channels and transporters ABCB10NM_012089, ABCB7 NM_004299, ABCD3 NM_002857, ABCE1 NM_002939, ABCF1NM_001090, ABCF2 NM_005692, ABCF3 NM_018358, CALM1[1][7] Calmodulingrasps calcium ions, MFSD11 NM_024311 similar to MSFD10 aka TETRAN ortetracycline transporter-like protein[1], MFSD12 NM_174983, MFSD3NM_138431, MFSD5 NM_032889, SLC15A4 NM_145648, SLC20A1 NM_005415,SLC25A11[1] mitochondrial oxoglutarate/malate carrier, SLC25A26NM_173471, SLC25A28 NM_031212, SLC25A3 NM_002635, SLC25A32 NM_030780,SLC25A38 NM_017875, SLC25A39 NM_016016, SLC25A44 NM_014655, SLC25A46NM_138773, SLC25A5 NM_001152, SLC27A4 NM_005094, SLC30A1 NM_021194,SLC30A5 NM_022902, SLC30A9 NM_006345, SLC35A2 NM_005660, SLC35A4NM_080670, SLC35B1 NM_005827, SLC35B2 NM_178148, SLC35C2 NM_015945,SLC35E1 NM_024881, SLC35E3 NM_018656, SLC35F5 NM_025181, SLC38A2NM_018976, SLC39A1 NM_014437, SLC39A3 NM_144564, SLC39A7 NM_006979,SLC41A3 NM_017836, SLC46A3 NM_181785, SLC48A1 NM_017842, the receptorsACVR1 NM_001105 similar to ACVRL1 TGF Beta receptor familyRendu-Osler-Weber syndrome, ACVR1B NM_004302, CD23[1] FCER2 low affinityIgE receptor (lectin); and the HLA/immunoglobulin/cell recognition groupBAT1 aka DDX39B which is involved in RNA splicing, BSG BasiginImmunoglobulin Superfamily, extracelluar metalloproteinase, MIFmacrophage migration inhibitory factor, and/or TAPBP [Wikipedia]. Insome embodiments, the housekeeping gene is an HLA type I, aG-protein-coupled receptor (GPCR), an ion channel or a receptor tyrosinekinase, preferably an HLA-A, HLA-B, HLA-C. In some embodiments, thehousekeeping gene is HLA-A. In some embodiments, the housekeeping geneis HLA-B. In some embodiments, the housekeeping gene is HLA-C.

Any relevant technology may be used to engineer a recognition moietythat confers to the aCARs and pCAR or iCARs specific binding to theirtargets. In some embodiments, the extracellular domain comprises (i) anantibody, derivative or fragment thereof, such as a humanized antibody;a human antibody; a functional fragment of an antibody; a single-domainantibody, such as a Nanobody; a recombinant antibody; and a single chainvariable fragment (ScFv); (ii) an antibody mimetic, such as an affibodymolecule; an affilin; an affimer; an affitin; an alphabody; ananticalin; an avimer; a DARPin; a fynomer; a Kunitz domain peptide; anda monobody; or (iii) an aptamer. Preferably, the extracellular domaincomprises an ScFv.

In some embodiments, the aCAR comprising an extracellular domain thatspecifically binds to a non-polymorphic cell surface epitope of anantigen or a single allelic variant of a polymorphic cell surfaceepitope. In some embodiments, the aCAR extracellular domain binds to anepitope that is a tumor-associated antigen epitope. In some embodiments,the aCAR extracellular domain binds to an epitope that is atumor-associated antigen is shared at least by cells of related tumorand normal tissue, and an intracellular domain comprising at least onesignal transduction element that activates and/or co-stimulates aneffector immune cell. In some embodiments, the aCAR used to treat thecancer is directed against or specifically binds to any membrane proteinwhich is expressed on the tumor tissue as long as the iCAR target isexpressed on every normal tissue in which the targeted aCAR protein isexpressed. In some embodiments, the aCAR is directed against orspecifically binds to, a non-polymorphic cell surface epitope selectedfrom but not limited to the following list of antigens: CD19, CD20,CD22, CD10, CD7, CD49f, CD56, CD74, CAIX Igκ, ROR1, ROR2, CD30, LewisY,CD33, CD34, CD38, CD123, CD28, CD44v6, CD44, CD41, CD133, CD138,NKG2D-L, CD139, BCMA, GD2, GD3, hTERT, FBP, EGP-2, EGP-40, FR-α, L1-CAM,ErbB2,3,4, EGFRvIII, VEGFR-2, IL-13Rα2, FAP, Mesothelin, c-MET, PSMA,CEA, kRas, MAGE-A1, MUC1MUC16, PDL1, PSCA, EpCAM, FSHR, AFP, AXL, CD80CD89, CDH17, CLD18, GPC3, TEM8, TGFB1, NY-ESO-1, WT-1 and EGFR In someembodiments, the aCAR binds to CD19. In some embodiments, the aCARdirected against or specifically binds to, a non-polymorphic cellsurface epitope of CD19.

In some embodiments, the iCAR is directed against or specifically bindsto a single allelic variant of an antigen not including the ephrinreceptors (e.g., EPHA 7) and claudins. In some embodiments, the iCAR isdirected against or specifically binds to an epitope encoded by a singleallelic variant of an HLA gene (HLA-A gene, HLA-B gene or HLA-C gene.

iii. Intracellular Domains: aCAR, iCAR and pCAR

The present invention also provides for intracellular domains as part ofthe aCAR, iCAR, and/or pCAR. In some embodiments, the intracellulardomain comprises at least one signal transduction element. In someembodiments, the intracellular domain comprises at least one signaltransduction element that inhibits an effector immune cell.

Generally, any relevant technology may be used to engineer a signaltransduction element that confers to the aCARs and pCAR or iCARs theability to induce a cellular function, including for example, theability to inhibit an effector immune cell or to activate orco-stimulate an effector immune cell.

In some embodiments, the at least one signal transduction element iscapable of inhibiting an effector immune cell. In some embodiments, theat least one signal transduction element capable of inhibiting aneffector immune cell is homologous to a signal transduction element ofan immune checkpoint protein. In some embodiments, the immune checkpointprotein is selected from the group consisting of PD1, CTLA4, BTLA, 2B4,CD160, CEACAM (including for example, CEACAM1), KIRs (including forexample KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1,KIR3DL2, KIR3DL3, LIR1, LIR2, LIR3, LIR5, LIR8 and CD94), NKG2A; LAG3;TIM3; V-domain Ig suppressor of T cell activation (VISTA); STimulator ofINterferon Genes (STING); immunoreceptor tyrosine-based inhibitory motif(ITIM)-containing proteins, T cell immunoglobulin and ITIM domain(TIGIT), and adenosine receptor (e.g. A2aR). In some embodiments, theimmune checkpoint protein is a negative immune regulator. In someembodiments, the negative immune regulator is selected from the groupconsisting of 2B4, LAG-3 and BTLA-4.

In some embodiments, the signal transduction element is capable ofactivating or co-stimulating an effector immune cell. In someembodiments, the signal transduction element is an activating domain. Insome embodiments, the signal transduction element is a co-stimulatorydomain. In some embodiments, the signal transduction element thatactivates or co-stimulates an effector immune cell is homologous to animmunoreceptor tyrosine-based activation motif (ITAM), an activatingkiller cell immunoglobulin-like receptor, or an adaptor molecule, and/ora co-stimulatory signal transduction element. In some embodiments, thesignal transduction element that activates or co-stimulates an effectorimmune cell is homologous to an immunoreceptor tyrosine-based activationmotif (ITAM). In some embodiments, the ITAM is from a protein includingbut not limited to CD3ζ or FcRγ chains. In some embodiments, the signaltransduction element that activates or co-stimulates an effector immunecell is homologous to an an activating killer cell immunoglobulin-likereceptor (KIR). In some embodiments, the MR includes, for example, butis not limited to KIR2DS and KIR3DS. In some embodiments, the signaltransduction element that activates or co-stimulates an effector immunecell is homologous to an adaptor molecule. In some embodiments, theadaptor molecule includes, for example, but is not limited to DAP12. Insome embodiments, the signal transduction element that activates orco-stimulates an effector immune cell is homologous to a co-stimulatorysignal transduction element. In some embodiments, the co-stimulatorysignal transduction element is from a protein including but not limitedto CD27, CD28, ICOS, CD137 (4-1BB), CD134 (OX40), and/or GITR. In someembodiments, the aCAR comprise a signal transduction element.

In some embodiments, the extracellular domain is fused through aflexible hinge and transmembrane canonic motif to said intracellulardomain.

In some embodiments, the use of a pCAR allows for uncoupling foruncoupling the activating moiety of the aCAR (FcRγ/CD3-ζ) from therecognition unit and the co-stimulatory element (e.g., CD28, 4-1BB). Insome embodiments, these elements are genetically placed on two differentpolypeptide products. In some embodiments, recoupling of these elements,which is mandatory for the aCAR function, will only take place by theaddition of a heterodimerizing drug which can bridge the respectivebinding sites incorporated onto each of the polypeptides separately.

Instead of an activating domain (such as FcRγ or CD3-ζ), an iCARpossesses a signaling domain derived from an inhibitory receptor whichcan antagonize T cell activation. In some embodiments, the iCARpossesses a signaling domain derived from an inhibitory receptor whichcan antagonize T cell activation. In some embodiments, the iCARsignaling domain is derived from an inhibitory receptor, including forexample but not limited to, a CTLA-4, a PD-1 or an NK inhibitoryreceptor.

iv. CAR-T Vector Construction (aCAR; iCAR; pCAR)

In some embodiments, the aCAR is encoded by a first nucleic acid vectorand the iCAR or pCAR is encoded by a second nucleic acid vector. In someembodiments, the aCAR is encoded by a first nucleic acid vector and theiCAR or pCAR is encoded by a second nucleic acid vector. In someembodiments, the aCAR is encoded by a first nucleic acid vector and theiCAR or pCAR is encoded by a second nucleic acid vector. In someembodiments, the the nucleotide sequence encoding for the iCAR or pCARis on a second vector.

In some embodiments, the present invention provides a vector comprisinga nucleic acid molecule of the invention as defined in any one of theabove embodiments, and at least one control element, such as a promoter,operably linked to the nucleic acid molecule.

In some embodiments, the vector is a lentiviral (LV) vector. In someembodiments, the LV vector is a commercially available LV vector. Insome embodiments, the LV vector includes but is not limited topLVX-Puro, pLVX-IRES-Puro/Neo/Hygro, pLVx-EF1a-IRES (TAKARA), and/orpcLV-EF1a (Sirion). In some embodiments, the LV vector is pLVX-Puro. Insome embodiments, the LV vector is pLVX-IRES-Puro/Neo/Hygro. In someembodiments, the LV vector is pLVx-EF1a-IRES (TAKARA). In someembodiments, the LV vector is pcLV-EF1a (Sirion).

In some embodiments, the vector comprises an EF1 promoter. In someembodiments, the vector comprises a CMV promoter. In some embodiments,the vector comprises an PGK promoter. In some embodiments, the vectorcomprises a CD8 hinge. In some embodiments, the vector comprises a CD28TM and 41BB costimulatory domain.

In some embodiments, the vector further comprises a nucleic acidmolecule comprising a nucleotide sequence encoding an aCAR comprising anextracellular domain specifically binding a non-polymorphic cell surfaceepitope of an antigen or a single allelic variant of a polymorphic cellsurface epitope, wherein said epitope is a tumor-associated antigen oris shared at least by cells of related tumor and normal tissue, and anintracellular domain comprising at least one signal transduction elementthat activates and/or co-stimulates an effector immune cell.

In some embodiments, the extracellular domain of the aCAR encoded by thenucleic acid comprised in the vector specifically binds to anon-polymorphic cell surface epitope of an antigen and the extracellulardomain of the iCAR specifically binds a single allelic variant of apolymorphic cell surface epitope of a different antigen than that towhich the extracellular domain of said aCAR binds.

In some embodiments, the extracellular domain of the iCAR encoded by thenucleic acid comprised in the vector, is directed against orspecifically binds to a single allelic variant of HLA genes, includingfor example, HLA-A gene, HLA-B gene or HLA-C gene; or against a singleallelic variant of a gene listed Table 8.

In some embodiments, the extracellular domain of the aCAR encoded by thenucleic acid comprised in the vector, is directed against orspecifically binds to, a non-polymorphic cell surface epitope selectedfrom the antigens listed in Table 1, such as CD19. In some embodiments,the aCAR target is any target with an extracellular domain.

In some embodiments, the extracellular domain of the iCAR encoded by thenucleic acid comprised in the vector, is directed against orspecifically binds to a single allelic variant of HLA genes, includingfor example, HLA-A gene, HLA-B gene or HLA-C gene or against a singleallelic variant of a gene listed Table 8; and the extracellular domainof the aCAR encoded by the nucleic acid comprised in the vector, isdirected against or specifically binds to, a non-polymorphic cellsurface epitope selected from the antigens listed in Table 1, such asCD19. In some embodiments, the aCAR target is any target with anextracellular domain.

In some embodiments, the at least one signal transduction element of theaCAR that activates or co-stimulates an effector immune cell ishomologous to an immunoreceptor tyrosine-based activation motif (ITAM)of for example CD3ζ or FcRγ chains; a transmembrane domain of anactivating killer cell immunoglobulin-like receptor (KIR) comprising apositively charged amino acid residue, or a positively charged sidechain or an activating KIR transmembrane domain of e.g., KIR2DS andKIR3DS, or an adaptor molecule such as DAP12; or a co-stimulatory signaltransduction element of for example CD27, CD28, ICOS, CD137 (4-1BB) orCD134 (OX40).

In some embodiments, the iCAR or pCAR is expressed by a first vector andthe aCAR is expressed by a second vector. In some embodiments, the iCARor pCAR and the aCAR are both expressed by the same vector.

In some embodiments, the nucleotide sequence of the vector comprises aninternal ribosome entry site (IRES) between the nucleotide sequenceencoding for the aCAR and the nucleotide sequence encoding for the iCAR.In general, the nucleotide sequence encoding for the aCAR and thenucleotide sequence encoding for the iCAR can be in any sequentialorder, but in particular embodiments, the nucleotide sequence encodingfor the aCAR is downstream of the nucleotide sequence encoding for theiCAR.

In some embodiments, the nucleotide sequences encoding for the aCAR iandthe iCAR are encoded on a single vector. In some embodiments, the vectorcomprises an internal ribosome entry site (IRES) between the nucleotidesequence encoding for the aCAR and the nucleotide sequence encoding forthe iCAR. In some embodiments, the nucleotide sequence encoding for theaCAR is downstream of the nucleotide sequence encoding for the iCAR. Insome embodiments, the nucleotide sequence comprises a viralself-cleaving 2A peptide located between the nucleotide sequenceencoding for the aCAR and the nucleotide sequence encoding for the iCAR.In some embodiments, the nucleotide sequence of the vector comprises aviral self-cleaving 2A peptide between the nucleotide sequence encodingfor the aCAR and the nucleotide sequence encoding for the iCAR. In someembodiments, the viral self-cleaving 2A peptide includes but is notlimited to T2A from Thosea asigna virus (TaV), F2A from Foot-and-mouthdisease virus (FMDV), E2A from Equine rhinitis A virus (ERAV) and/or P2Afrom Porcine teschovirus-1 (PTV1). In some embodiments, the viralself-cleaving 2A peptide is T2A from Thosea asigna virus (TaV). In someembodiments, the viral self-cleaving 2A peptide is F2A fromFoot-and-mouth disease virus (FMDV). In some embodiments, the viralself-cleaving 2A peptide is E2A from Equine rhinitis A virus (ERAV). Insome embodiments, the viral self-cleaving 2A peptide is P2A from Porcineteschovirus-1 (PTV1).

In some embodiments, the vector comprises a nucleotide sequence encodingthe constitutive aCAR linked via a flexible linker to said iCAR.

The immune cells may be transfected with the appropriate nucleic acidmolecule described herein by e.g., RNA transfection or by incorporationin a plasmid fit for replication and/or transcription in a eukaryoticcell or a viral vector. In some embodiments, the vector is selected froma retroviral or lentiviral vector.

Combinations of retroviral vector and an appropriate packaging line canalso be used, where the capsid proteins will be functional for infectinghuman cells. Several amphotropic virus-producing cell lines are known,including PA12 (Miller, et al. (1985) Mol. Cell. Biol 5:431-437); PA317(Miller, et al. (1986) Mol. Cell. Bioi. 6:2895-2902); and CRIP (Danos,et ai. (1988) Proc. Nati. Acad. Sci. USA 85:6460-6464). Alternatively,non-amphotropic particles can be used, such as, particles pseudotypedwith VSVG, RD 114 or GAL V envelope. Cells can further be transduced bydirect co-culture with producer cells, e.g., by the method of Bregni, etai. (1992) Blood 80: 1418-1422, or culturing with viral supernatantalone or concentrated vector stocks, e.g., by the method of Xu, et ai.(1994) Exp. Hemat. 22:223-230; and Hughes, et ai. (1992) J Clin. Invest.89: 1817.

In another aspect, the present invention provides a method of preparingan inhibitory chimeric antigen receptor (iCAR) capable of preventing orattenuating undesired activation of an effector immune cell, accordingto the present invention as defined above, the method comprising: (i)retrieving a list of human genomic variants of protein-encoding genesfrom at least one database of known variants; (ii) filtering the list ofvariants retrieved in (i) by: (a) selecting variants resulting in anamino acid sequence variation in the protein encoded by the respectivegene as compared with its corresponding reference allele, (b) selectingvariants of genes wherein the amino acid sequence variation is in anextracellular domain of the encoded protein, (c) selecting variants ofgenes that undergo loss of heterozygosity (LOH) at least in one tumor,and (d) selecting variants of genes that are expressed at least in atissue of origin of the at least one tumor in which they undergo LOHaccording to (c), thereby obtaining a list of variants having an aminoacid sequence variation in an extracellular domain in the proteinencoded by the respective gene lost in the at least one tumor due to LOHand expressed at least in a tissue of origin of the at least one tumor;(iii) defining a sequence region comprising at least one single variantfrom the list obtained in (ii), sub-cloning and expressing the sequenceregion comprising the at least one single variant and a sequence regioncomprising the corresponding reference allele thereby obtaining therespective epitope peptides; (iv) selecting an iCAR binding domain,which specifically binds either to the epitope peptide encoded by thecloned sequence region, or to the epitope peptide encoded by thecorresponding reference allele, obtained in (iii); and (vii) preparingiCARs as defined herein above, each comprising an iCAR binding domain asdefined in (iv).

In some embodiments, the candidate variants of genes that are selectedundergo LOH in at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 100% in a certain tumor type.

In some embodiments, the minor allele frequency for each variantselected equals or exceeds 1, 2, 3, 4 or 5% in at least one population.

In another aspect, the present invention is directed to a combination oftwo or more nucleic acid molecules, each one comprising a nucleotidesequence encoding a different member of a controlled effector immunecell activating system, said nucleic acid molecules being part of orforming a single continues nucleic acid molecule, or comprising two ormore separate nucleic acid molecules, wherein the controlled effectorimmune activating system directs effector immune cells to kill tumorcells that have lost one or more chromosomes or fractions thereof due toLoss of Heterozygosity (LOH) and spares cells of related normal tissue,and wherein (a) the first member comprises an activating chimericantigen receptor (aCAR) polypeptide comprising a first extracellulardomain that specifically binds to a non-polymorphic cell surface epitopeof an antigen or to a single allelic variant of a different polymorphiccell surface epitope and said non-polymorphic or polymorphic cellsurface epitope is a tumor-associated antigen or is shared by cells ofrelated abnormal and normal mammalian tissue; and (b) the second membercomprises a regulatory polypeptide comprising a second extracellulardomain that specifically binds to a single allelic variant of apolymorphic cell surface epitope not expressed by an abnormal mammaliantissue due to LOH but present on all cells of related mammalian normaltissue.

In some embodiments, the first member is selected from: (a) aconstitutive aCAR further comprising an intracellular domain comprisingat least one signal transduction element that activates and/orco-stimulates an effector immune cell; and (b) a conditional aCARfurther comprising an intracellular domain comprising a first member ofa binding site for a heterodimerizing small molecule and optionally atleast one co-stimulatory signal transduction element, but lacking anactivating signal transduction element; and the second member is: (c) aninhibiting chimeric antigen receptor (iCAR) further comprising anintracellular domain comprising at least one signal transduction elementthat inhibits an effector immune cell; or (d) a protective chimericantigen receptor (pCAR) further comprising an extracellular regulatoryregion comprising a substrate for a sheddase; a transmembrane canonicmotif comprising a substrate for an intramembrane-cleaving protease; andan intracellular domain, said intracellular domain comprising at leastone signal transduction element that activates and/or co-stimulates aneffector immune cell and a second member of a binding site for aheterodimerizing small molecule.

In some embodiments (i) the extracellular domain of the iCAR or pCARspecifically binds a single allelic variant of a polymorphic cellsurface epitope of an antigen, which is a different antigen than that towhich the extracellular domain of the aCAR binds; (ii) the extracellulardomain of said pCAR or iCAR specifically binds a single allelic variantof a different polymorphic cell surface epitope of the same antigen towhich the extracellular domain of said aCAR binds; or (iii) theextracellular domain of said pCAR or iCAR specifically binds a differentsingle allelic variant of the same polymorphic cell surface epitope towhich the extracellular domain of said aCAR binds.

In some pCAR embodiments, the substrate for a sheddase is a substratefor a disintegrin and metalloproteinase (ADAM) or a beta-secretase 1(BACE1). In some embodiments, the substrate forms part of theextracellular domain and comprises Lin 12/Notch repeats and an ADAMprotease cleavage site.

It is generally accepted that there is no consistent sequence motifpredicting ADAM cleavage, but Caescu et al. (Caescu et al., 2009)disclose in Table 3 a large number of ADAM10 and/or ADAM17 substratesequences, which are hereby incorporated by reference as if fullydisclosed herein, and which may serve as a substrate for ADAM in thepCAR of the present invention. In some embodiments, the ADAM substratesequences are those of amyloid precursor protein, BTC, CD23, Collagen,DII-1, Ebola glycoprotein, E-cadherin, EGF, Epiregulin, Fas Ligand,growth hormone receptor, HB-EGF, type II interleukin-1 receptor, IL-6receptor, L-selectin, N-cadherin, Notch, p55 TNF receptor, p75 TNFreceptor, Pme117, Prion protein, receptor-type protein tyrosinephosphatase Z, TGF-α, TNF or TR (Caescu et al., 2009).

It may be advantageous to use an ADAM10 cleavage sequence in the pCAR ofthe present invention because ADAM 10 is constitutively present atcomparably high levels on e.g., lymphocytes. In contrast to ADAM10, theclose relative TACE/ADAM17 is detected at only low levels onunstimulated cells. ADAM17 surface expression on T cell blasts israpidly induced by stimulation (Ebsen et al., 2013).

Hemming et al. (Hemming et al., 2009) report that no consistent sequencemotif predicting BACE1 cleavage has been identified in substrates versusnon-substrates, but discloses in Table 1 a large number of BACE1substrates having BAC1 cleavage sequences, which are hereby incorporatedby reference as if fully disclosed herein, and which may serve as asubstrate for BACE1 in the pCAR of the present invention.

In some pCAR embodiments, the substrate for an intramembrane-cleavingprotease is a substrate for an SP2, a γ-secretase, a signal peptidepeptidase (spp), a spp-like protease or a rhomboid protease.

Rawson et al. (Rawson, 2013) disclose that SP2 substrates have at leastone type 2 membrane-spanning helix and include a helix-destabilizingmotif, such as an Asp-Pro motif in a SP2 substrate. This paper disclosesin Table 1 a number of SP2 substrates having SP2-cleavage sequences,which are hereby incorporated by reference as if fully disclosed herein,and which may serve as a substrate for SP2 in the pCAR of the presentinvention.

Haapasalo and Kovacs (Haapasalo and Kovacs, 2011) teach that amyloid-βprotein precursor (AβPP) is a substrate for presenilin (PS)-dependentγ-secretase (PS/γ-secretase), and that at least 90 additional proteinshave been found to undergo similar proteolysis by this enzyme complex.γ-secretase substrates have some common features: most substrateproteins are type-I transmembrane proteins; the PS/γ-secretase-mediatedγ-like cleavage (corresponding to the &-cleavage in AβPP, which releasesAICD) takes place at or near the boundary of the transmembrane andcytoplasmic domains. The &-like cleavage site flanks a stretch ofhydrophobic amino acid sequence rich in lysine and/or arginine residues.It appears that PS/γ-secretase cleavage is not dependent on a specificamino acid target sequence at or adjacent to the cleavage site, butrather perhaps on the conformational state of the transmembrane domain.

Haapasalo and Kovacs disclose in Table 1 a list of γ-secretasesubstrates, the cleavage sequences of which are hereby incorporated byreference as if fully disclosed herein, and which may serve as asubstrate for γ-secretases in the pCAR of the present invention.

Voss et al. (Voss et al., 2013) teach that so far no consensus cleavagesite based on primary sequence elements within the substrate has beendescribed for GxGD aspartyl proteases (spps). Transmembrane domains ofmembrane proteins preferentially adopt an α-helical confirmation inwhich their peptide bonds are hardly accessible to proteases. In orderto make transmembrane domains susceptible for intramembrane proteolysisit was therefore postulated that their α-helical content needs to bereduced by helix destabilizing amino acids. Consistent with thishypothesis, various signal peptides have been shown to contain helixdestabilizing amino acids within their h-region which criticallyinfluence their proteolytic processing by SPP. In addition, polarresidues within the h-region of signal peptides may influence cleavageby SPP, as for instance serine and cysteine residues within the signalpeptide of various HCV strains are critical for SPP cleavage. Whetherthese polar residues also simply affect the helical content of thesignal peptides or the hydroxyl or sulfhydryl group in particular isrequired to trigger cleavage by SPP is not yet fully understood.Similarly, cleavage of the Bri2 transmembrane domain by SPPL2b issignificantly increased when the α-helical content of the Bri2transmembrane domainis reduced. Interestingly, only one amino acidresidue out of four residues with a putative helix destabilizing potencysignificantly reduced the α-helical content of the Bri2 transmembranedomainin a phospholipid-based environment. This suggests thatdestabilization of an α-helical transmembrane domain is not simplycaused by certain amino acid residues but that rather context andposition of these amino acids determine their helix destabilizingpotential and thus the accessibility of transmembrane domains tointramembrane proteolysis by SPP/SPPLs. Voss et al. further disclose inTable 1 a list of spp and spp-like substrates, the cleavage sequences ofwhich are hereby incorporated by reference as if fully disclosed herein,and which may serve as a substrate for spp in the pCAR of the presentinvention.

Bergbold et al. (Bergbold and Lemberg, 2013) teach that for rhomboidproteases, two different models for substrate recognition have beensuggested. In the first model, the conformational flexibility of thesubstrate peptide backbone combined with immersion of the membrane inthe vicinity of the rhomboid active site is sufficient to providespecificity. For the well-characterized Drosophila substrate Spitz, aglycine-alanine motif has been shown to serve as a helix break thatallows unfolding of the transmembrane domain into the rhomboid activesite. The second model suggests that rhomboid proteases primarilyrecognize a specific sequence surrounding the cleavage site, and thattransmembrane helix-destabilizing residues are a secondary featurerequired for some substrates only. The specific sequence has not yetbeen identified. Bergbold et al. disclose in Table 3 a list of rhomboidprotease substrates, the cleavage sequences of which are herebyincorporated by reference as if fully disclosed herein, and which mayserve as a substrate for rhomboid proteases in the pCAR of the presentinvention.

In view of the above, since in most cases no consensus motif has yetbeen established for the intramembrane-cleaving proteases, and sinceassays for identifying intramembrane-cleaving protease substrates arewell known in the art as described in literature cited herein above, thepCAR may comprise an amino acid sequence identified as such and mayfurther comprise transmembrane helix-destabilizing residues.

In some embodiments, the substrate forms part of the transmembranecanonic motif and is homologous to/derived from a transmembrane domainof Notch, ErbB4, E-cadherin, N-cadherin, ephrin-B2, amyloid precursorprotein or CD44.

In some embodiments, the comprises a nucleotide sequence encoding anextracellular domain and an intracellular domain of said conditionalaCAR as separate proteins, wherein each domain is independently fused toa transmembrane canonic motif and comprises a different member of abinding site for a heterodimerizing small molecule.

In some embodiments, the each one of the first and second member of thebinding site for a heterodimerizing small molecule is derived from aprotein selected from: (i) Tacrolimus (FK506) binding protein (FKBP) andFKBP; (ii) FKBP and calcineurin catalytic subunit A (CnA); (iii) FKBPand cyclophilin; (iv) FKBP and FKBP-rapamycin associated protein (FRB);(v) gyrase B (GyrB) and GyrB; (vi) dihydrofolate reductase (DHFR) andDHFR; (vii) DmrB homodimerization domain (DmrB) and DmrB; (viii) a PYLprotein (a.k.a. abscisic acid receptor and as RCAR) and ABI; and (ix)GAI Arabidopsis thaliana protein (a.k.a Gibberellic Acid Insensitive andDELLA protein GAI; GAI) and GID1 Arabidopsis thaliana protein (alsoknown as Gibberellin receptor GID1; GID1).

v. Construction of Effector Cells

In still another aspect, the present invention provides a method forpreparing a safe effector immune cell comprising: (i) transfecting aTCR-engineered effector immune cell directed to a tumor-associatedantigen with a nucleic acid molecule comprising a nucleotide sequenceencoding an iCAR or pCAR as defined herein above or transducing thecells with a vector or (ii) transfecting a naïve effector immune cellwith a nucleic acid molecule comprising a nucleotide sequence encodingan iCAR or pCAR as defined herein above and a nucleic acid moleculecomprising a nucleotide sequence encoding an aCAR as defined hereinabove; or transducing an effector immune cell with a vector as definedherein above.

In some embodiments, the immune cell for use in engineering includes butis not limited to a T-cell, a natural killer cell, or a cytokine-inducedkiller cell. In some embodiments, the immune cell for use in engineeringincludes but is not limited to a Jurkat T-cell, a Jurkat-NFAT T-cell,and/or a peripheral blood mononuclear cell (PBMC).

In yet another aspect, the present invention provides a safe effectorimmune cell obtained by the method of the present invention as describedabove. The safe effector immune cell may be a redirected T cellexpressing an exogenous T cell receptor (TCR) and an iCAR or pCAR,wherein the exogenous TCR is directed to a non-polymorphic cell surfaceepitope of an antigen or a single allelic variant of a polymorphic cellsurface epitope, wherein said epitope is a tumor-associated antigen oris shared at least by cells of related tumor and normal tissue, and theiCAR or pCAR is as defined above; or the safe effector immune cell is aredirected effector immune cell such as a natural killer cell or a Tcell expressing an iCAR or pCAR and an aCAR as defined above.

In some embodiments, the safe effector immune cell, expresses on itssurface an aCAR comprising an extracellular domain that specificallybinds to a non-polymorphic cell surface epitope of an antigen and aniCAR or pCAR comprising an extracellular domain that specifically bindsa single allelic variant of a polymorphic cell surface epitope of adifferent antigen to which the extracellular domain of said aCAR binds.In some embodiments, the extracellular domain of the iCAR or pCARspecifically binds a single allelic variant of a different polymorphiccell surface epitope are of the same antigen to which the extracellulardomain of said aCAR binds; or the extracellular domain of the iCAR orpCAR specifically binds a different single allelic variant of the samepolymorphic cell surface epitope area to which the extracellular domainof said aCAR binds.

In some embodiments, the extracellular domain of the aCAR expressed onthe cell surface specifically binds to a non-polymorphic cell surfaceepitope selected from the antigens listed in Table 1, such as CD19. Insome embodiments, the target is any target with an extracellular domain.

In some embodiments, the extracellular domain of the iCAR and or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of an HLA-A, HLA-B, HLA-C, HLA-G, HLA-E,HLA-F, HLA-K, HLA-L, HLA-DM, HLA-DO, HLA-DP, HLA_DQ, or HLA-DR gene oragainst a single allelic variant of a gene listed Table 8.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of an HLA-A gene, HLA-B gene or HLA-C geneor against a single allelic variant of a gene listed Table 8; and theextracellular domain of the aCAR expressed on the cell surface isdirected against or specifically binds to, a non-polymorphic cellsurface epitope selected from the antigens listed in Table 1, such as,for example, but not limited to, CD19. In some embodiments, the aCARtarget is any target with an extracellular domain.

In some embodiments, the aCAR and the iCAR are present on the cellsurface as separate proteins.

In some embodiments, the expression level on the cell surface of thenucleotide sequence encoding the iCAR is greater than or equal to theexpression level of the nucleotide sequence encoding the aCAR.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of an at least one extracellular polymorphicepitope.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ABCA4, ADAM30, AQP10, ASTN1, C1orf101, CACNA1S, CATSPER4, CD101,CD164L2, CD1A, CD1C, CD244, CD34, CD46, CELSR2, CHRNB2, CLCA2, CLDN19,CLSTN1, CR1, CR2, CRB1, CSF3R, CSMD2, ECE1, ELTD1, EMC1, EPHA10, EPHA2,EPHA8, ERMAP, FCAMR, FCER1A, FCGR1B, FCGR2A, FCGR2B, FCGR3A, FCRL1,FCRL3, FCRL4, FCRL5, FCRL6, GJB4, GPA33, GPR157, GPR37L1, GPR88, HCRTR1,IGSF3, IGSF9, IL22RA1, IL23R, ITGA10, KIAA1324, KIAA2013, LDLRAD2, LEPR,LGR6, LRIG2, LRP8, LRRC52, LRRC8B, LRRN2, LY9, MIA3, MR1, MUC1, MXRA8,NCSTN, NFASC, NOTCH2, NPR1, NTRK1, OPN3, OR10J1, OR10J4, OR10K1, OR1OR2,OR10T2, OR10X1, OR11L1, OR14A16, OR14I1, OR14K1, OR2AK2, OR2C3, OR2G2,OR2G3, OR2L2, OR2M7, OR2T12, OR2T27, OR2T1, OR2T3, OR2T29, OR2T33,OR2T34, OR2T35, OR2T3, OR2T4, OR2T5, OR2T6, OR2T7, OR2T8, OR2W3, OR6F1,OR6K2, OR6K3, OR6K6, OR6N1, OR6P1, OR6Y1, PDPN, PEAR1, PIGR, PLXNA2,PTCH2, PTCHD2, PTGFRN, PTPRC, PTPRF, PTGFRN, PVRL4, RHBG, RXFP4, S1PR1,SCNN1D, SDC3, SELE, SELL, SELP, SEMA4A, SEMA6C, SLAMF7, SLAMF9, SLC2A7,SLC5A9, TACSTD2, TAS1R2, TIE1, TLR5, TMEM81, TNFRSF14, TNFRSF1B,TRABD2B, USH2A, VCAM1, and ZP4.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ABCG5, ALK, ASPRV1, ATRAID, CD207, CD8B, CHRNG, CLEC4F, CNTNAP5,CRIM1, CXCR1, DNER, DPP10, EDAR, EPCAM, GPR113, GPR148, GPR35, GPR39,GYPC, IL1RL1, ITGA4, ITGA6, ITGAV, LCT, LHCGR, LRP1B, LRP2, LY75, MARCO,MERTK, NRP2, OR6B2, PLA2R1, PLB1, PROKR1, PROM2, SCN7A, SDC1, SLC23A3,SLC5A6, TGOLN2, THSD7B, TM4SF20, TMEFF2, TMEM178A, TPO, and TRABD2A.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ACKR2, ALCAM, ANO10, ATP13A4, BTLA, CACNA1D, CACNA2D2, CACNA2D3,CASR, CCRL2, CD200, CD200R1, CD86, CD96, CDCP1, CDHR4, CELSR3, CHL1,CLDN11, CLDN18, CLSTN2, CSPG5, CX3CR1, CXCR6, CYP8B1, DCBLD2, DRD3,EPHA6, EPHB3, GABRR3, GP5, GPR128, GPR15, GPR27, GRM2, GRM7, HEG1,HTR3C, HTR3D, HTR3E, IGSF11, IL17RC, IL17RD, IL17RE, IL5RA, IMPG2,ITGA9, ITGB5, KCNMB3, LRIG1, LRRC15, LRRN1, MST1R, NAALADL2, NRROS,OR5AC1, OR5H1, OR5H14, OR5H15, OR5H6, OR5K2, OR5K3, OR5K4, PIGX, PLXNB1,PLXND1, PRRT3, PTPRG, ROBO2, RYK, SEMA5B, SIDT1, SLC22A14, SLC33A1,SLC4A7, SLITRK3, STAB1, SUSD5, TFRC, TLR9, TMEM108, TMEM44, TMPRSS7,TNFSF10, UPK1B, VIPR1, and ZPLD1.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ANTXR2, BTC, CNGA1, CORIN, EGF, EMCN, ENPEP, EPHA5, ERVMER34-1, EVC2,FAT1, FAT4, FGFRL1, FRAS1, GPR125, GRID2, GYPA, GYPB, KDR, KIAA0922,KLB, MFSD8, PARM1, PDGFRA, RNF150, TENM3, TLR10, TLR1, TLR6, TMEM156,TMPRSS11A, TMPRSS11B, TMPRSS11E, TMPRSS11F, UGT2A1, and UNC5C.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ADAM19, ADRB2, BTNL3, BTNL8, BTNL9, C5orf15, CATSPER3, CD180, CDH12,CDHR2, COL23A1, CSF1R, F2RL2, FAM174A, FAT2, FGFR4, FLT4, GABRA6,GABRG2, GPR151, GPR98, GRM6, HAVCR1, HAVCR2, IL31RA, IL6ST, IL7R,IQGAP2, ITGA1, ITGA2, KCNMB1, LIFR, LNPEP, MEGF10, NIPAL4, NPR3, NRG2,OR2V1, OR2Y1, OSMR, PCDH12, PCDH1, PCDHA1, PCDHA2, PCDHA4, PCDHA8,PCDHA9, PCDHB10, PCDHB11, PCDHB13, PCDHB14, PCDHB15, PCDHB16, PCDHB2,PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHGA1, PCDHGA4, PDGFRB, PRLR, SEMA5A,SEMA6A, SGCD, SLC1A3, SLC22A4, SLC22A5, SLC23A1, SLC36A3, SLC45A2,SLC6A18, SLC6A19, SLCO6A1, SV2C, TENM2, TIMD4, and UGT3A1.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof BAI3, BTN1A1, BTN2A1, BTN2A2, BTN3A1, BTN3A2, BTNL2, CD83, DCBLD1,DLL1, DPCR1, ENPP1, ENPP3, ENPP4, EPHA7, GABBR1, GABRR1, GCNT6, GFRAL,GJB7, GLP1R, GPR110, GPR111, GPR116, GPR126, GPR63, GPRC6A, HFE, HLA-A,HLA-B, HLA-C, HLA-DOA, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1,HLA-DQB2, HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-G, IL20RA, ITPR3,KIAA0319, LMBRD1, LRFN2, LRP11, MAS1L, MEP1A, MICA, MICB, MOG, MUC21,MUC22, NCR2, NOTCH4, OPRM1, OR10C1, OR12D2, OR12D3, OR14J1, OR2B2,OR2B6, OR2J1, OR2W1, OR5V1, PDE10A, PI16, PKHD1, PTCRA, PTK7, RAET1E,RAET1G, ROS1, SDIM1, SLC16A10, SLC22A1, SLC44A4, TAAR2, TREM1, TREML1,and TREML2.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof AQP1, C7orf50, CD36, CDHR3, CNTNAP2, DPP6, EGFR, EPHA1, EPHB6,ERVW-1, GHRHR, GJC3, GPNMB, GRM8, HUS1, HYAL4, KIAA1324L, LRRN3, MET,MUC12, MUC17, NPC1L1, NPSR1, OR2A12, OR2A14, OR2A25, OR2A42, OR2A7,OR2A2, OR2AE1, OR2F2, OR6V1, PILRA, PILRB, PKD1L1, PLXNA4, PODXL,PTPRN2, PTPRZ1, RAMP3, SLC29A4, SMO, TAS2R16, TAS2R40, TAS2R4, TFR2,THSD7A, TMEM213, TTYH3, ZAN, and ZP3.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ADAM18, ADAM28, ADAM32, ADAM7, ADAM9, ADRA1A, CDH17, CHRNA2, CSMD1,CSMD3, DCSTAMP, FZD6, GPR124, NRG1, OR4F21, PKHD1L1, PRSS55, SCARA3,SCARA5, SDC2, SLC10A5, SLC39A14, SLC39A4, SLCO5A1, TNFRSF10A, andTNFRSF10B.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ABCA1, AQP7, ASTN2, C9orf135, CA9, CD72, CNTNAP3B, CNTNAP3, CRB2,ENTPD8, GPR144, GRIN3A, IZUMO3, KIAA1161, MAMDC4, MEGF9, MUSK, NOTCH1,OR13C2, OR13C3, OR13C5, OR13C8, OR13C9, OR13D1, OR13F1, OR1B1, OR1J2,OR1K1, OR1L1, OR1L3, OR1L6, OR1L8, OR1N1, OR1N2, OR1Q1, OR2S2, PCSK5,PDCD1LG2, PLGRKT, PTPRD, ROR2, SEMA4D, SLC31A1, TEK, TLR4, TMEM2, andVLDLR.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ABCC2, ADAMS, ADRB1, ANTXRL, ATRNL1, C10orf54, CDH23, CDHR1, CNNM2,COL13A1, COL17A1, ENTPD1, FZD8, FGFR2, GPR158, GRID1, IL15RA, IL2RA,ITGA8, ITGB1, MRC1, NRG3, NPFFR1, NRP1, OPN4, PCDH15, PKD2L1, PLXDC2,PRLHR, RET, RGR, SLC16A9, SLC29A3, SLC39A12, TACR2, TCTN3, TSPAN15,UNC5B, and VSTM4.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof AMICA1, ANO1, ANO3, APLP2, C11orf24, CCKBR, CD248, CD44, CD5, CD6,CD82, CDON, CLMP, CRTAM, DCHS1, DSCAML1, FAT3, FOLH1, GDPD4, GDPD5,GRIK4, HEPHL1, HTR3B, IFITM10, IL10RA, KIRREL3, LGR4, LRP4, LRP5,LRRC32, MCAM, MFRP, MMP26, MPEG1, MRGPRE, MRGPRF, MRGPRX2, MRGPRX3,MRGPRX4, MS4A4A, MS4A6A, MTNR1B, MUC15, NAALAD2, NAALADL1, NCAM1, NRXN2,OR10A2, OR10A5, OR10A6, OR10D3, OR10G4, OR10G7, OR10G8, OR10G9, OR10Q1,OR10S1, OR1S1, OR2AG1, OR2AG2, OR2D2, OR4A47, OR4A15, OR4A5, OR4C11,OR4C13, OR4C15, OR4C16, OR4C3, OR4C46, OR4C5, OR4D6, OR4A8P, OR4D9,OR4S2, OR4X1, OR51E1, OR51L1, OR52A1, OR52E1, OR52E2, OR52E4, OR52E6,OR52I1, OR52I2, OR52J3, OR52L1, OR52N1, OR52N2, OR52N4, OR52W1, OR56B1,OR56B4, OR5A1, OR5A2, OR5AK2, OR5AR1, OR5B17, OR5B3, OR5D14, OR5D16,OR5D18, OR5F1, OR5I1, OR5L2, OR5M11, OR5M3, OR5P2, OR5R1, OR5T2, OR5T3,OR5W2, OR6A2, OR6T1, OR6X1, OR8A1, OR8B12, OR8B2, OR8B3, OR8B4, OR8D1,OR8D2, OR8H1, OR8H2, OR8H3, OR8I2, OR8J1, OR8J2, OR8J3, OR8K1, OR8K3,OR8K5, OR8U1, OR9G1, OR9G4, OR9Q2, P2RX3, PTPRJ, ROBO3, SIGIRR,SLC22A10, SLC3A2, SLC5A12, SLCO2B1, SORL1, ST14, SYT8, TENM4, TMEM123,TMEM225, TMPRSS4, TMPRSS5, TRIM5, TRPM5, TSPAN18, and ZP1.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ANO4, AVPR1A, BCL2L14, CACNA2D4, CD163, CD163L1, CD27, CD4, CLEC12A,CLEC1B, CLEC2A, CLEC4C, CLEC7A, CLECL1, CLSTN3, GPR133, GPRC5D, ITGA7,ITGB7, KLRB1, KLRC2, KLRC3, KLRC4, KLRF1, KLRF2, LRP1, LRP6, MANSC1,MANSC4, OLR1, OR10AD1, OR10P1, OR2AP1, OR6C1, OR6C2, OR6C3, OR6C4,OR6C6, OR6C74, OR6C76, OR8S1, OR9K2, ORAI1, P2RX4, P2RX7, PRR4, PTPRB,PTPRQ, PTPRR, SCNN1A, SELPLG, SLC2A14, SLC38A4, SLC5A8, SLC6A15, SLC8B1,SLCO1A2, SLCO1B1, SLCO1B7, SLCO1C1, SSPN, STAB2, TAS2R10, TAS2R13,TAS2R14, TAS2R20, TAS2R30, TAS2R31, TAS2R42, TAS2R43, TAS2R46, TAS2R7,TMEM119, TMEM132B, TMEM132C, TMEM132D, TMPRSS12, TNFRSF1A, TSPAN8, andVSIG10.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ATP4B, ATP7B, FLT3, FREM2, HTR2A, KL, PCDH8, RXFP2, SGCG, SHISA2,SLC15A1, SLITRK6, and TNFRSF19.

In some embodiments, the recognition moiety for use in the aCAR, iCARand/or pCAR provides specifity to at least one extracellular polymorphicepitope in a gene product from a gene selected from the group consistingof ADAM21, BDKRB2, C14orf37, CLEC14A, DLK1, FLRT2, GPR135, GPR137C,JAG2, LTB4R2, MMP14, OR11G2, OR11H12, OR11H6, OR4K1, OR4K15, OR4K5,OR4L1, OR4N2, OR4N5, SLC24A4, and SYNDIG1L.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ANPEP, CD276, CHRNA7, CHRNB4, CSPG4, DUOX1, DUOX2, FAM174B, GLDN,IGDCC4, ITGA11, LCTL, LTK, LYSMD4, MEGF11, NOX5, NRG4, OCA2, OR4F4,OR4M2, OR4N4, PRTG, RHCG, SCAMPS, SEMA4B, SEMA6D, SLC24A1, SLC24A5,SLC28A1, SPG11, STRA6, TRPM1, and TYRO3.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ATP2C2, CACNA1H, CD19, CDH11, CDH15, CDH16, CDH3, CDH5, CNGB1,CNTNAP4, GDPD3, GPR56, GPR97, IFT140, IL4R, ITFG3, ITGAL, ITGAM, ITGAX,KCNG4, MMP15, MSLNL, NOMO1, NOMO3, OR2C1, PIEZO1, PKD1, PKD1L2, QPRT,SCNN1B, SEZ6L2, SLC22A31, SLC5A11, SLC7A6, SPN, TMC5, TMC7, TMEM204,TMEM219, and TMEM8A.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ABCC3, ACE, AOC3, ARL17B, ASGR2, C17orf80, CD300A, CD300C, CD300E,CD300LF, CD300LG, CHRNB1, CLEC10A, CNTNAP1, CPD, CXCL16, ERBB2,FAM171A2, GCGR, GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B, ITGA3, ITGAE,ITGB3, KCNJ12, LRRC37A2, LRRC37A3, LRRC37A, LRRC37B, MRC2, NGFR, OR1A2,OR1D2, OR1G1, OR3A1, OR3A2, OR4D1, OR4D2, RNF43, SCARF1, SCN4A, SDK2,SECTM1, SEZ6, SHPK, SLC26A11, SLC5A10, SPACA3, TMEM102, TMEM132E,TNFSF12, TRPV3, TTYH2, and TUSC5.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof APCDD1, CDH19, CDH20, CDH7, COLEC12, DCC, DSC1, DSG1, DSG3, DYNAP,MEP1B, PTPRM, SIGLEC15, and TNFRSF11A.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ABCA7, ACPT, BCAM, C19orf38, C19orf59, C5AR1, CATSPERD, CATSPERG,CD22, CD320, CD33, CD97, CEACAM19, CEACAM1, CEACAM21, CEACAM3, CEACAM4,CLEC4M, DLL3, EMR1, EMR2, EMR3, ERVV-1, ERVV-2, FAM187B, FCAR, FFAR3,FPR1, FXYD5, GFY, GP6, GPR42, GRIN3B, ICAM3, IGFLR1, IL12RB1, IL27RA,KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KIRREL2, KISS1R,LAIR1, LDLR, LILRA1, LILRA2, LILRA4, LILRA6, LILRB1, LILRB2, LILRB3,LILRB4, LILRB5, LINGO3, LPHN1, LRP3, MADCAM1, MAG, MEGF8, MUC16, NCR1,NOTCH3, NPHS1, OR10H1, OR10H2, OR10H3, OR10H4, OR1I1, OR2Z1, OR7A10,OR7C1, OR7D4, OR7E24, OR7G1, OR7G2, OR7G3, PLVAP, PTGIR, PTPRH, PTPRS,PVR, SCN1B, SHISA7, SIGLEC10, SIGLEC11, SIGLEC12, SIGLEC5, SIGLEC6,SIGLEC8, SIGLEC9, SLC44A2, SLC5A5, SLC7A9, SPINT2, TARM1, TGFBR3L, TMC4,TMEM91, TMEM161A, TMPRSS9, TNFSF14, TNFSF9, TRPM4, VN1R2, VSIG10L,VSTM2B, and ZNRF4.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ABHD12, ADAM33, ADRA1D, APMAP, ATRN, CD40, CD93, CDH22, CDH26, CDH4,FLRT3, GCNT7, GGT7, JAG1, LRRN4, NPBWR2, OCSTAMP, PTPRA, PTPRT, SEL1L2,SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3, SLC2A10, SLC4A11, SSTR4, andTHBD.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof CLDN8, DSCAM, ICOSLG, IFNAR1, IFNGR2, IGSF5, ITGB2, KCNJ15, NCAM2,SLC19A1, TMPRSS15, TMPRSS2, TMPRSS3, TRPM2, and UMODL1.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof CACNA1I, CELSR1, COMT, CSF2RB, GGT1, GGT5, IL2RB, KREMEN1, MCHR1,OR11H1, P2RX6, PKDREJ, PLXNB2, SCARF2, SEZ6L, SSTR3, SUSD2, TMPRSS6, andTNFRSF13C.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of a gene selected from the group consistingof ATP6AP2, ATP7A, CNGA2, EDA2R, FMR1NB, GLRA4, GPR112, GUCY2F, HEPH,P2RY10, P2RY4, PLXNA3, PLXNB3, TLR8, VSIG4, and XG.

In some embodiments, the extracellular domain of the iCAR and/or pCARexpressed on the cell surface is directed against or specifically bindsto a single allelic variant of HLA-A2. In some embodiments, theextracellular domain of the iCAR and/or pCAR expressed on the cellsurface is directed against or specifically binds to a single allelicvariant of CD20. In some embodiments, the iCAR will be directed towardHLA-A2. In some embodiments, the iCAR will be directed toward CD20. Insome embodiments, the aCAR with be directed toward CD19. In someembodiments, the iCAR/aCAR set will be HLA-A2 and CD19 respectively. Insome embodiments, the iCAR/aCAR set will include CD20 and CD19respectively.

vi. Preparation of Target Cells

In some embodiments, the target cells are prepared and tested in an invitro system. In some embodiments, an in vitro recombinant system willbe established for testing the functionality of the iCAR and/or pCARconstructs in inhibiting the activity of the aCAR towards the off-targetcells. In some embodiments, target cells expressing the aCAR epitope,iCAR epitope or both will be produced. In some embodiments, target cellsexpressing the aCAR epitope, pCAR epitope or both will be produced. Insome embodiments, the recombinant cells expressing the aCAR epitope willrepresent the on-target ‘on-tumor’ cells while the cells expressing bothaCAR and iCAR epitopes would represent the on target ‘off-tumor’ healthycells.

In some embodiments, the iCAR/aCAR set will be HLA-A2 and CD19respectively, recombinant cells expressing HLA-A2, CD19 or both will beproduced by transfecting cell line (e.g., Hela, Hela-Luciferase or Raji)with expression vector coding for these genes. For detection ofrecombinant CD19 and HLA-A2 expression, both genes will be fused to aprotein tag (e.g., HA or Flag or Myc etc). In some embodiments, theiCAR/aCAR target set will be CD20/CD19 and the recombinant cells willexpress CD19, CD20 or both.

In some embodiments, the expression vector comprising the iCAR/aCARtarget set is transfected into a cell. In some embodiments, theexpression vector is transfected into a cell to produce the target andoff-tumor effects.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ABCA4, ADAM30, AQP10, ASTN1, C1orf101,CACNA1S, CATSPER4, CD101, CD164L2, CD1A, CD1C, CD244, CD34, CD46,CELSR2, CHRNB2, CLCA2, CLDN19, CLSTN1, CR1, CR2, CRB1, CSF3R, CSMD2,ECE1, ELTD1, EMC1, EPHA10, EPHA2, EPHA8, ERMAP, FCAMR, FCER1A, FCGR1B,FCGR2A, FCGR2B, FCGR3A, FCRL1, FCRL3, FCRL4, FCRL5, FCRL6, GJB4, GPA33,GPR157, GPR37L1, GPR88, HCRTR1, IGSF3, IGSF9, IL22RA1, IL23R, ITGA10,KIAA1324, KIAA2013, LDLRAD2, LEPR, LGR6, LRIG2, LRP8, LRRC52, LRRC8B,LRRN2, LY9, MIA3, MR1, MUC1, MXRA8, NCSTN, NFASC, NOTCH2, NPR1, NTRK1,OPN3, OR10J1, OR10J4, OR10K1, OR1OR2, OR10T2, OR10X1, OR11L1, OR14A16,OR14I1, OR14K1, OR2AK2, OR2C3, OR2G2, OR2G3, OR2L2, OR2M7, OR2T12,OR2T27, OR2T1, OR2T3, OR2T29, OR2T33, OR2T34, OR2T35, OR2T3, OR2T4,OR2T5, OR2T6, OR2T7, OR2T8, OR2W3, OR6F1, OR6K2, OR6K3, OR6K6, OR6N1,OR6P1, OR6Y1, PDPN, PEAR1, PIGR, PLXNA2, PTCH2, PTCHD2, PTGFRN, PTPRC,PTPRF, PTGFRN, PVRL4, RHBG, RXFP4, S1PR1, SCNN1D, SDC3, SELE, SELL,SELP, SEMA4A, SEMA6C, SLAMF7, SLAMF9, SLC2A7, SLC5A9, TACSTD2, TAS1R2,TIE1, TLR5, TMEM81, TNFRSF14, TNFRSF1B, TRABD2B, USH2A, VCAM1, and ZP4.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ABCG5, ALK, ASPRV1, ATRAID, CD207, CD8B,CHRNG, CLEC4F, CNTNAP5, CRIM1, CXCR1, DNER, DPP10, EDAR, EPCAM, GPR113,GPR148, GPR35, GPR39, GYPC, IL1RL1, ITGA4, ITGA6, ITGAV, LCT, LHCGR,LRP1B, LRP2, LY75, MARCO, MERTK, NRP2, OR6B2, PLA2R1, PLB1, PROKR1,PROM2, SCN7A, SDC1, SLC23A3, SLC5A6, TGOLN2, THSD7B, TM4SF20, TMEFF2,TMEM178A, TPO, and TRABD2AD2A.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ACKR2, ALCAM, ANO10, ATP13A4, BTLA,CACNA1D, CACNA2D2, CACNA2D3, CASR, CCRL2, CD200, CD200R1, CD86, CD96,CDCP1, CDHR4, CELSR3, CHL1, CLDN11, CLDN18, CLSTN2, CSPG5, CX3CR1,CXCR6, CYP8B1, DCBLD2, DRD3, EPHA6, EPHB3, GABRR3, GP5, GPR128, GPR15,GPR27, GRM2, GRM7, HEG1, HTR3C, HTR3D, HTR3E, IGSF11, IL17RC, IL17RD,IL17RE, IL5RA, IMPG2, ITGA9, ITGB5, KCNMB3, LRIG1, LRRC15, LRRN1, MST1R,NAALADL2, NRROS, OR5AC1, OR5H1, OR5H14, OR5H15, OR5H6, OR5K2, OR5K3,OR5K4, PIGX, PLXNB1, PLXND1, PRRT3, PTPRG, ROBO2, RYK, SEMA5B, SIDT1,SLC22A14, SLC33A1, SLC4A7, SLITRK3, STAB1, SUSD5, TFRC, TLR9, TMEM108,TMEM44, TMPRSS7, TNFSF10, UPK1B, VIPR1, and ZPLD1.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ANTXR2, BTC, CNGA1, CORIN, EGF, EMCN,ENPEP, EPHA5, ERVMER34-1, EVC2, FAT1, FAT4, FGFRL1, FRAS1, GPR125,GRID2, GYPA, GYPB, KDR, KIAA0922, KLB, MFSD8, PARM1, PDGFRA, RNF150,TENM3, TLR10, TLR1, TLR6, TMEM156, TMPRSS11A, TMPRSS11B, TMPRSS11E,TMPRSS11F, UGT2A1, and UNC5C.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ADAM19, ADRB2, BTNL3, BTNL8, BTNL9,C5orf15, CATSPER3, CD180, CDH12, CDHR2, COL23A1, CSF1R, F2RL2, FAM174A,FAT2, FGFR4, FLT4, GABRA6, GABRG2, GPR151, GPR98, GRM6, HAVCR1, HAVCR2,IL31RA, IL6ST, IL7R, IQGAP2, ITGA1, ITGA2, KCNMB1, LIFR, LNPEP, MEGF10,NIPAL4, NPR3, NRG2, OR2V1, OR2Y1, OSMR, PCDH12, PCDH1, PCDHA1, PCDHA2,PCDHA4, PCDHA8, PCDHA9, PCDHB10, PCDHB11, PCDHB13, PCDHB14, PCDHB15,PCDHB16, PCDHB2, PCDHB3, PCDHB4, PCDHB5, PCDHB6, PCDHGA1, PCDHGA4,PDGFRB, PRLR, SEMA5A, SEMA6A, SGCD, SLC1A3, SLC22A4, SLC22A5, SLC23A1,SLC36A3, SLC45A2, SLC6A18, SLC6A19, SLCO6A1, SV2C, TENM2, TIMD4, andUGT3A1.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of BAI3, BTN1A1, BTN2A1, BTN2A2, BTN3A1,BTN3A2, BTNL2, CD83, DCBLD1, DLL1, DPCR1, ENPP1, ENPP3, ENPP4, EPHA7,GABBR1, GABRR1, GCNT6, GFRAL, GJB7, GLP1R, GPR110, GPR111, GPR116,GPR126, GPR63, GPRC6A, HFE, HLA-A, HLA-B, HLA-C, HLA-DOA, HLA-DPA1,HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRB1, HLA-DRB5,HLA-E, HLA-F, HLA-G, IL20RA, ITPR3, KIAA0319, LMBRD1, LRFN2, LRP11,MAS1L, MEP1A, MICA, MICB, MOG, MUC21, MUC22, NCR2, NOTCH4, OPRM1,OR10C1, OR12D2, OR12D3, OR14J1, OR2B2, OR2B6, OR2J1, OR2W1, OR5V1,PDE10A, PI16, PKHD1, PTCRA, PTK7, RAET1E, RAET1G, ROS1, SDIM1, SLC16A10,SLC22A1, SLC44A4, TAAR2, TREM1, TREML1, and TREML2.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of AQP1, C7orf50, CD36, CDHR3, CNTNAP2, DPP6,EGFR, EPHA1, EPHB6, ERVW-1, GHRHR, GJC3, GPNMB, GRM8, HUS1, HYAL4,KIAA1324L, LRRN3, MET, MUC12, MUC17, NPC1L1, NPSR1, OR2A12, OR2A14,OR2A25, OR2A42, OR2A7, OR2A2, OR2AE1, OR2F2, OR6V1, PILRA, PILRB,PKD1L1, PLXNA4, PODXL, PTPRN2, PTPRZ1, RAMP3, SLC29A4, SMO, TAS2R16,TAS2R40, TAS2R4, TFR2, THSD7A, TMEM213, TTYH3, ZAN, and ZP3.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ADAM18, ADAM28, ADAM32, ADAM7, ADAM9,ADRA1A, CDH17, CHRNA2, CSMD1, CSMD3, DCSTAMP, FZD6, GPR124, NRG1,OR4F21, PKHD1L1, PRSS55, SCARA3, SCARA5, SDC2, SLC10A5, SLC39A14,SLC39A4, SLCO5A1, TNFRSF10A, and TNFRSF10B.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ABCA1, AQP7, ASTN2, C9orf135, CA9, CD72,CNTNAP3B, CNTNAP3, CRB2, ENTPD8, GPR144, GRIN3A, IZUMO3, KIAA1161,MAMDC4, MEGF9, MUSK, NOTCH1, OR13C2, OR13C3, OR13C5, OR13C8, OR13C9,OR13D1, OR13F1, OR1B1, OR1J2, OR1K1, OR1L1, OR1L3, OR1L6, OR1L8, OR1N1,OR1N2, OR1Q1, OR2S2, PCSK5, PDCD1LG2, PLGRKT, PTPRD, ROR2, SEMA4D,SLC31A1, TEK, TLR4, TMEM2, and VLDLR.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ABCC2, ADAMS, ADRB1, ANTXRL, ATRNL1,C10orf54, CDH23, CDHR1, CNNM2, COL13A1, COL17A1, ENTPD1, FZD8, FGFR2,GPR158, GRID1, IL15RA, IL2RA, ITGA8, ITGB1, MRC1, NRG3, NPFFR1, NRP1,OPN4, PCDH15, PKD2L1, PLXDC2, PRLHR, RET, RGR, SLC16A9, SLC29A3,SLC39A12, TACR2, TCTN3, TSPAN15, UNC5B, and VSTM4.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of AMICA1, ANO1, ANO3, APLP2, C11orf24, CCKBR,CD248, CD44, CD5, CD6, CD82, CDON, CLMP, CRTAM, DCHS1, DSCAML1, FAT3,FOLH1, GDPD4, GDPD5, GRIK4, HEPHL1, HTR3B, IFITM10, IL10RA, KIRREL3,LGR4, LRP4, LRP5, LRRC32, MCAM, MFRP, MMP26, MPEG1, MRGPRE, MRGPRF,MRGPRX2, MRGPRX3, MRGPRX4, MS4A4A, MS4A6A, MTNR1B, MUC15, NAALAD2,NAALADL1, NCAM1, NRXN2, OR10A2, OR10A5, OR10A6, OR10D3, OR10G4, OR10G7,OR10G8, OR10G9, OR10Q1, OR10S1, OR1S1, OR2AG1, OR2AG2, OR2D2, OR4A47,OR4A15, OR4A5, OR4C11, OR4C13, OR4C15, OR4C16, OR4C3, OR4C46, OR4C5,OR4D6, OR4A8P, OR4D9, OR4S2, OR4X1, OR51E1, OR51L1, OR52A1, OR52E1,OR52E2, OR52E4, OR52E6, OR52I1, OR52I2, OR52J3, OR52L1, OR52N1, OR52N2,OR52N4, OR52W1, OR56B1, OR56B4, OR5A1, OR5A2, OR5AK2, OR5AR1, OR5B17,OR5B3, OR5D14, OR5D16, OR5D18, OR5F1, OR5I1, OR5L2, OR5M11, OR5M3,OR5P2, OR5R1, OR5T2, OR5T3, OR5W2, OR6A2, OR6T1, OR6X1, OR8A1, OR8B12,OR8B2, OR8B3, OR8B4, OR8D1, OR8D2, OR8H1, OR8H2, OR8H3, OR8I2, OR8J1,OR8J2, OR8J3, OR8K1, OR8K3, OR8K5, OR8U1, OR9G1, OR9G4, OR9Q2, P2RX3,PTPRJ, ROBO3, SIGIRR, SLC22A10, SLC3A2, SLC5A12, SLCO2B1, SORL1, ST14,SYT8, TENM4, TMEM123, TMEM225, TMPRSS4, TMPRSS5, TRIM5, TRPM5, TSPAN18,and ZP1.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ANO4, AVPR1A, BCL2L14, CACNA2D4, CD163,CD163L1, CD27, CD4, CLEC12A, CLEC1B, CLEC2A, CLEC4C, CLEC7A, CLECL1,CLSTN3, GPR133, GPRC5D, ITGA7, ITGB7, KLRB1, KLRC2, KLRC3, KLRC4, KLRF1,KLRF2, LRP1, LRP6, MANSC1, MANSC4, OLR1, OR10AD1, OR10P1, OR2AP1, OR6C1,OR6C2, OR6C3, OR6C4, OR6C6, OR6C74, OR6C76, OR8S1, OR9K2, ORAI1, P2RX4,P2RX7, PRR4, PTPRB, PTPRQ, PTPRR, SCNN1A, SELPLG, SLC2A14, SLC38A4,SLC5A8, SLC6A15, SLC8B1, SLCO1A2, SLCO1B1, SLCO1B7, SLCO1C1, SSPN,STAB2, TAS2R10, TAS2R13, TAS2R14, TAS2R20, TAS2R30, TAS2R31, TAS2R42,TAS2R43, TAS2R46, TAS2R7, TMEM119, TMEM132B, TMEM132C, TMEM132D,TMPRSS12, TNFRSF1A, TSPAN8, and VSIG10.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ATP4B, ATP7B, FLT3, FREM2, HTR2A, KL,PCDH8, RXFP2, SGCG, SHISA2, SLC15A1, SLITRK6, and TNFRSF19.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ADAM21, BDKRB2, C14orf37, CLEC14A, DLK1,FLRT2, GPR135, GPR137C, JAG2, LTB4R2, MMP14, OR11G2, OR11H12, OR11H6,OR4K1, OR4K15, OR4K5, OR4L1, OR4N2, OR4N5, SLC24A4, and SYNDIG1L.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ANPEP, CD276, CHRNA7, CHRNB4, CSPG4, DUOX1,DUOX2, FAM174B, GLDN, IGDCC4, ITGA11, LCTL, LTK, LYSMD4, MEGF11, NOX5,NRG4, OCA2, OR4F4, OR4M2, OR4N4, PRTG, RHCG, SCAMPS, SEMA4B, SEMA6D,SLC24A1, SLC24A5, SLC28A1, SPG11, STRA6, TRPM1, and TYRO3.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ATP2C2, CACNA1H, CD19, CDH11, CDH15, CDH16,CDH3, CDH5, CNGB1, CNTNAP4, GDPD3, GPR56, GPR97, IFT140, IL4R, ITFG3,ITGAL, ITGAM, ITGAX, KCNG4, MMP15, MSLNL, NOMO1, NOMO3, OR2C1, PIEZO1,PKD1, PKD1L2, QPRT, SCNN1B, SEZ6L2, SLC22A31, SLC5A11, SLC7A6, SPN,TMC5, TMC7, TMEM204, TMEM219, and TMEM8A.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ABCC3, ACE, AOC3, ARL17B, ASGR2, C17orf80,CD300A, CD300C, CD300E, CD300LF, CD300LG, CHRNB1, CLEC10A, CNTNAP1, CPD,CXCL16, ERBB2, FAM171A2, GCGR, GLP2R, GP1BA, GPR142, GUCY2D, ITGA2B,ITGA3, ITGAE, ITGB3, KCNJ12, LRRC37A2, LRRC37A3, LRRC37A, LRRC37B, MRC2,NGFR, OR1A2, OR1D2, OR1G1, OR3A1, OR3A2, OR4D1, OR4D2, RNF43, SCARF1,SCN4A, SDK2, SECTM1, SEZ6, SHPK, SLC26A11, SLC5A10, SPACA3, TMEM102,TMEM132E, TNFSF12, TRPV3, TTYH2, and TUSC5.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of APCDD1, CDH19, CDH20, CDH7, COLEC12, DCC,DSC1, DSG1, DSG3, DYNAP, MEP1B, PTPRM, SIGLEC15, and TNFRSF11A.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ABCA7, ACPT, BCAM, C19orf38, C19orf59,C5AR1, CATSPERD, CATSPERG, CD22, CD320, CD33, CD97, CEACAM19, CEACAM1,CEACAM21, CEACAM3, CEACAM4, CLEC4M, DLL3, EMR1, EMR2, EMR3, ERVV-1,ERVV-2, FAM187B, FCAR, FFAR3, FPR1, FXYD5, GFY, GP6, GPR42, GRIN3B,ICAM3, IGFLR1, IL12RB1, IL27RA, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1,KIR3DL2, KIR3DL3, KIRREL2, KISS1R, LAIR1, LDLR, LILRA1, LILRA2, LILRA4,LILRA6, LILRB1, LILRB2, LILRB3, LILRB4, LILRB5, LINGO3, LPHN1, LRP3,MADCAM1, MAG, MEGF8, MUC16, NCR1, NOTCH3, NPHS1, OR10H1, OR10H2, OR10H3,OR10H4, OR1I1, OR2Z1, OR7A10, OR7C1, OR7D4, OR7E24, OR7G1, OR7G2, OR7G3,PLVAP, PTGIR, PTPRH, PTPRS, PVR, SCN1B, SHISA7, SIGLEC10, SIGLEC11,SIGLEC12, SIGLEC5, SIGLEC6, SIGLEC8, SIGLEC9, SLC44A2, SLC5A5, SLC7A9,SPINT2, TARM1, TGFBR3L, TMC4, TMEM91, TMEM161A, TMPRSS9, TNFSF14,TNFSF9, TRPM4, VN1R2, VSIG10L, VSTM2B, and ZNRF4.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ABHD12, ADAM33, ADRA1D, APMAP, ATRN, CD40,CD93, CDH22, CDH26, CDH4, FLRT3, GCNT7, GGT7, JAG1, LRRN4, NPBWR2,OCSTAMP, PTPRA, PTPRT, SEL1L2, SIGLEC1, SIRPA, SIRPB1, SIRPG, SLC24A3,SLC2A10, SLC4A11, SSTR4, and THBD.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of CLDN8, DSCAM, ICOSLG, IFNAR1, IFNGR2,IGSF5, ITGB2, KCNJ15, NCAM2, SLC19A1, TMPRSS15, TMPRSS2, TMPRSS3, TRPM2,and UMODL1.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of CACNA1I, CELSR1, COMT, CSF2RB, GGT1, GGT5,IL2RB, KREMEN1, MCHR1, OR11H1, P2RX6, PKDREJ, PLXNB2, SCARF2, SEZ6L,SSTR3, SUSD2, TMPRSS6, and TNFRSF13C.

In some embodiments, the expression vector codes for a gene selectedfrom the group consisting of ATP6AP2, ATP7A, CNGA2, EDA2R, FMR1NB,GLRA4, GPR112, GUCY2F, HEPH, P2RY10, P2RY4, PLXNA3, PLXNB3, TLR8, VSIG4,and XG.

In some embodiments, the safe effector immune cells used for treatingcancer as defined above express on their surface an aCAR comprising anextracellular domain that specifically binds to a tumor-associatedantigen or a cell surface epitope of an antigen and an iCAR comprisingan extracellular domain that specifically binds a single allelic variantof a polymorphic cell surface epitope of an antigen expressed at leastin a tissue of origin of the tumor, such as any of those listed above,which is a different antigen than that to which the extracellular domainof said aCAR binds. In some embodiments, the iCAR is expressed in thesame tissue as the aCAR is expressed in. In some embodiments, the aCARand iCAR are different alleles of the same gene. In some embodiments,the aCAR and iCAR are different proteins, and hence are differentalleles.

A. In Vitro Assays

In some embodiments, the iCAR and/or pCAR will be tested for activity ineffects, including effectiveness and ability to inhibit, using a varietyof assays. In some embodiments, the inhibitory effect of the iCAR and/orpCAR will be tested in-vitro and/or in-vivo. In some embodiments, theinhibitory effect of the iCAR and/or pCAR will be tested in-vitro. Insome embodiments, the inhibitory effect of the iCAR and/or pCAR will betested in-vivo. In some embodiments, the in vitro assays measurecytokine secretion and/or cytotoxicity effects. In some embodiments, thein vivo assays will evaluate the iCAR and/or pCAR inhibition andprotection to on-target off tumor xenografts. In some embodiments, thethe in vivo assays will evaluate the iCAR and/or pCAR inhibition andprotection to on-target off tumor tissue and/or viral organs.

i. Luciferase Cytotoxicity Assay

In some embodiments, the iCAR and/or pCAR are evaluated using aluciferase cytotoxicity assay. Generally, for a luciferase cytotoxicassay, recombinant target cells (which can be referred to as “T”) areengineered to express firefly luciferase. In some embodiments,commercial Hela-Luc cells can be transfected with DNA coding for thetarget proteins. The in vitro luciferase assay can be performedaccording to the Bright-Glo Luciferase assay (commercially availablefrom Promega or BPS Biosciences or other commercial vendors). Transducedeffector (E) T cells (which have been transduced with both iCAR or pCARand aCAR or aCAR or mock CAR) can be incubated for 24-48 hrs withrecombinant target cells expressing HLA-A2, CD19 or both CD19 andHLA-A2, or CD20, or both CD20 and CD19 to be tested in differenteffector to target ratios. In some embodiments, the iCAR/aCAR orpCAR/aCAR pair comprises any of aCAR, pCAR and/or iCAR with thecomponents as described above. In some embodiments, the iCAR/aCAR paircomprises an HLA-A2 targeted iCAR and a CD19 targeted aCAR. In someembodiments, the iCAR/aCAR pair comprises a CD20 targeted iCAR and aCD19 targeted aCAR. Cell killing will be quantified indirectly byestimating the number of live cells with the Bright-Glo Luciferasesystem.

In some embodiments, the ‘off-tumor’ cytotoxicity can be optimized bysorting transduced T cell populations according to iCAR/aCAR expressionlevel or by selecting sub population of recombinant target cellsaccording to their target expression, including for example, expressionof the gene product encoding for at least one extracellular polymorphicepitope. In some embodiments, the aCAR, iCAR, and/or pCAR target is anytarget with an extracellular domain. In some embodiments, the sorting isbased on CD19 or HLA-A2 expression level.

In some embodiments, the iCAR and/or pCAR is examined to determinewhether the iCAR transduced T cells can discriminate between the‘on-tumor’ cells (e.g., tumor cells) and ‘off-tumor’ cells (e.g.,non-tumor cells) in vitro. Generally, this is tested by examining thekilling effect of transduced T cells incubated with a mix of ‘on-tumor’and ‘off-tumor’ cells at a ratio of 1:1. In some embodiments, the ratiois 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, or 1:8. The on tumor recombinant cellscan be distinguished from the ‘off-tumor’ recombinant cells byluciferase expression in embodiments where only one cell population willbe engineered to express the luciferase gene at a time). Killing can bequantified after 24-48 hrs of co-incubation using the Bright-GloLuciferase assay (Promega).

In some embodiments, the iCAR/aCAR and/or pCAR/aCAR transduced T cellsexhibit about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, and/or about 95% less off-tumorcell killing as compared to T cells transduced with aCAR but nottransduced with the iCAR and/or pCAR. In some embodiments, the iCAR/aCARand/or pCAR/aCAR transduced T cells exhibit about 1-fold, about 2-fold,about 3-fold, about 4-fold, about 5-fold, or about 10-fold lessoff-tumor cell killing as compared to T cells transduced with aCAR butnot transduced with the iCAR and/or pCAR.

ii. Caspase 3

In some embodiments, caspase 3-detection assays are employed to examinethe iCAR and/or pCAR to determine the level of apoptic of the ‘on-tumor’cells (e.g., tumor cells) and ‘off-tumor’ cells (e.g., non-tumor cells)in vitro. In some embodiments, caspase_3-detection of cytotoxiclymphocyte (CTL) induced apoptosis by an antibody to activated cleavedcaspase 3 is examined.

Generally, one of the pathways by which CTLs kill target cells is byinducing apoptosis through the Fas ligand. The CASP3 protein is a memberof the cysteine-aspartic acid protease (caspase) family. Typically,sequential activation of caspases plays a significant role in theexecution-phase of cell apoptosis and as such, cleavage of pro-caspase 3to caspase 3 results in conformational change and expression ofcatalytic activity. The cleaved activated form of caspase 3 can berecognized specifically by a monoclonal antibody.

In some embodiments, transduced T cells can be incubated with either‘on-tumor’ (e.g., mimicking tumor) and ‘off-tumor’ cells (e.g.,mimicking non-tumor) recombinant cells. In some embodiments, the‘on-tumor’ (e.g., tumor) and ‘off-tumor’ cells (e.g., non-tumor)recombinant cells have been previously labeled with CFSE((5(6)-Carboxyfluorescein N-hydroxysuccinimidyl ester)) or other celltracer dye (e.g., CellTrace Violet). In some embodiments, co-incubationof target cells with effector cells occurs for about 1 hour to 6 abouthours, about 2 hours to about 5 hours, or about 2 to about 4 hrs. Insome embodiments, target cell apoptosis is quantified by flow cytometry.Cells can be permeabilized and fixed by an inside staining kit (Miltenyior BD bioscience) and stained with an antibody for activated caspase 3(BD bioscience).

In some embodiments, the iCAR/aCAR and/or pCAR/aCAR transduced T cellsinduce about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, and/or about 95% less off-tumor cellapoptosis as compared to T cells transduced with aCAR but not transducedwith the iCAR and/or pCAR. In some embodiments, the aCAR/iCAR and/oraCAR/pCAR transduced T cells induce about 1-fold, about 2-fold, about3-fold, about 4-fold, about 5-fold, or about 10-fold less off-tumor cellapoptosis as compared to T cells transduced with aCAR but not transducedwith the iCAR and/or pCAR.

iii. Time-Lapse Microscopy

Time Lapse Micros CTL—

Time lapse microscopy of the iCAR and/or pCAR transduced T cells can beemployed in order to discern target binding. In some embodiments, targetcells will be labeled with a reporter gene (for example but not limitedto a fluorescent proten such as mCherry). In some embodiments,transduced T cells are incubated with either ‘on-tumor’ or ‘off-tumor’cells for up to 5 days. In some embodiments, time lapse microscopy canbe used to visualize killing. In some embodiments, flow cytometryanalysis using viable cell number staining and CountBright beads(Invitrogen) for determining target cell number at end-point time willbe conducted.

In some embodiments, in order to determine if the aCAR/iCAR or aCAR/pCARtransduced T cells can discern targets in vitro, each recombinant targetcells (‘on-tumor’ or ‘off-tumor’) is labeled with a different reporterprotein (for example GFP and mCherry). In some embodiments, any reportprotein pair would work, so long as the reporter pair contains tworeporters which are easily distinguishable. In some embodiments,transduced T cells (Effector cells) will be co-incubated with therecombinant cells (target cells) at a 1:1 ratio of E/T. In someembodiments, the ration of effector to target (E/T) includes but is notlimited to 16:1, 12:1, 10:1, 8:1, 6:1, 4:1, 2:1, or 1:1. In someembodiments, the cell fate is then examined by microscopy imaging.

iv. Cytokine Release

Cytokine release can be examined in order to determine T cellsactivation. In some embodiments, iCAR/aCAR and/or pCAR/aCAR transduced Tcells are incubated with the recombinant target cells and cytokineproduction for one or more cytokines is quantified, for example, eitherby measuring cytokine secretion in cell culture supernatant according toBioLegend's ELISA MAX™ Deluxe Set kit or by flow cytometry analysis ofthe percentage of T cells producing cytokines. For the flow cytometryanalysis, a Golgi stop is generally employed to prevent the secretion ofthe cytokines. In some embodiments, following a 6 hour and 18 hour to 24hour incubation of the transduced T cells with target cells, T cellswill be permeabilized and fixed by an inside staining kit (Miltenyi) andstained with antibodies for the T cell markers (CD3 and CD8) and for oneor more cytokines. In some embodiments, the cytokines include but arenot limited to IL-2, INFγ, and/or TNFα.

v. CD107a Staining

Staining for CD107a can also be examined in order to determine cytolyticactivity of the transduced T cells. Generally, degranulating of T cellscan be identified by the surface expression of CD107a, a lysosomalassociated membrane protein (LAMP-1), and surface expression of LAMP-1has been shown to correlate with CD8 T cell cytotoxicity. Further, thismolecule is located on the luminal side of lysosomes. Typically, uponactivation, CD107a is transferred to the cell membrane surface ofactivated lymphocytes. Moreover, CD107a is expressed on the cell surfacetransiently and is rapidly re-internalized via the endocytic pathway.Therefore, while not being bound by theory, CD107a detection ismaximized by antibody staining during cell stimulation and by theaddition of monensin (for example, to prevent acidification andsubsequent degradation of endocytosed CD107a antibody complexes).

In some embodiments, the aCAR/iCAR and/or aCAR/pCAR transducedtransduced T cells are incubated with the target cells for about 6 oursto about 24 hrs and CD107a expression on the CD8 T cells is examined. Insome embodiments, the target cells expresso only one target proteinrecognized by aCAR (as in tumor cells) or target cells expressing bothtarget proteins recognized by aCAR and iCAR (as in normal cells). Insome embodiments, the iCAR and/or pCAR transduced transduced T cells areincubated with the target cells for about 6 ours to about 24 hrs in thepresence of monensin and CD107a expression on the CD8 T cells isfollowed by flow cytometry using conjugated antibodies against the Tcell surface markers (for example, CD3 and CD8) and a conjugatedantibody for CD107a.

vi. Quantitation of Secreted Cytokines by ELISA

In some embodiments, following co-cultivation of transduced T-cells(Jurkat, or primary T-cells) expressing iCAR or aCAR or both aCAR andiCAR with modified target cells, expressing iCAR or aCAR or both aCARand iCAR antigens on their cell surface, conditioned medium will becollected, and cytokine's concentration will be measured by cytokineELISA. In some embodiments, the cytokine is selected from the groupconsisting of IL-2, INFγ and/or TNFα. In some embodiments, the cytokineis selected from the group consisting of IL-2. In some embodiments, thecytokine is selected from the group consisting of INFγ. In someembodiments, the cytokine is selected from the group consisting of TNFα.In some embodiments, a decrease of about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or about 99% is demonstrated with dual CAR (aCAR/iCAR) transduced cells.

vii. Cytokines Secretion Measured by Cytometric Bead Array (CBA) Assay

Cytometric Bead Array (CBA) is used to measure a variety of soluble andintracellular proteins, including cytokines, chemokines and growthfactors. In some embodiments, T-cells (primary T-cells or Jurkat cells)transduced with aCAR or both aCAR and iCAR constructs (Effector cells)are stimulated with modified target cells expressing both iCAR and aCARor aCAR or iCAR target antigens on their cell surface. In someembodiments, the effector to target ratio ranges from 20:1 up to 1:1. Insome embodiments, the effector to target ratio ranges from 20:1, 19:1,18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1,2:1, or 1:1. In some embodiments, following severalhours of co-incubation the effector cells produce and secrete cytokineswhich indicate their effector state. In some embodiments, thesupernatant of the reaction is collected, and secreted IL-2 was measuredand quantified by multiplex CBA assay.

In some embodiments, a decrease of about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or about 99% is demonstrated with dual CAR (aCAR/iCAR) transduced cellswere co-incubated with target cells expressing both target antigens ascompared to IL-2 secretion resulted from co-incubation of the sameeffector cells with target cells expressing only one target. In someembodiments, a decrease of about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%in IL-2 secretion was demonstrated when dual CAR (aCAR/iCAR) transducedcells were co-incubated with target cells expressing both targetantigens as compared to IL-2 secretion resulted from co-incubation ofthe same effector cells with target cells expressing only one target. Insome embodiments, a decrease of 86%. In some embodiments, the aCAR is aCD19 aCAR. In some embodiments, the iCAR is an HLA-A2 iCAR. In someembodiments, the iCAR is a CD20 iCAR. In some embodiments, the aCAR/iCARpair is CD19 aCAR and HLA-A2 iCAR. In some embodiments, the aCAR/iCARpair is CD19 aCAR and a CD20 iCAR

viii. T-Cell Degranulation Assay as Measured by CD107a Staining

In some embodiments, degranulating of T cells can be identified by thesurface expression of CD107a, a lysosomal associated membrane protein(LAMP-1). In some embodiments, surface expression of LAMP-1 has beenshown to correlate with CD8 T cell cytotoxicity. In some embodiments,granulation (CD107a) is a marker for killing potential.

B. In Vivo Assays

In some embodiments, the iCAR/aCAR and/or iCAR/pCAR pairs are tested foreffectiveness in vivo. In some embodiments, NOD/SCID/γc- or similar miceare inoculated intravenously with tumor cells. In some embodiments, thetumor cells are CD19 positive NALM 6 (ATCC, human B-ALL cell line) cellsthat are engineered to express firefly luciferase. In some embodiments,for establishment of and/or differentiation between ‘on-target’ cellsand ‘off-tumor’ cells, NALM 6 can be engineered to express the iCARand/or pCAR epitope thereby representing the healthy cells. In someembodiments, the iCAR and/or pCAR epitope comprises at least oneextracellular polymorphic epitope. In some embodiments, the iCAR and/orpCAR epitope is from HLA-A2 or CD20. Other cells that could be employedin these assays include but are not limited to Raji or any otherrecombinant cell lines. In some embodiments, such assays can be in a PDX(patient derived xenograft) model.

For the assay, mice will be divided into study groups; one group will beinjected with the NALM 6 cells while the other will be injected with theNALM-6 expressing the iCAR epitope. Several days later, mice will beinfused intravenously with T cells transduced with aCAR, aCAR/iCAR and acontrol group of untransduced T cells or no T cells. Mice will besacrificed and tumor burden will be quantified according to total flux.

According to one embodiment of the assay, in order to test whether the Tcells expressing the iCAR and/or pCAR construct could discriminatebetween the target cells and off target cells in vivo within the sameorganism, mice are injected with a 1:1 mixture of the‘on-tumor’/′off-tumor NALM-6 cells, followed by injection of transducedT cells expressing either the aCAR alone or both aCAR and iCAR. Withthis embodiment, upon sacrifice of the mice the presence of the‘on-tumor’ and ‘off-tumor cells in the spleen and bone marrow will beanalyzed by flow cytometry for the two markers, CD19 and the iCARepitope.

i. In Vivo CTL Assay in Human Xenograft Mouse Models

In some embodiments, to test whether T-cells expressing both aCAR andiCAR constructs discriminate between the target cells and ‘off-target’cells within the same organism and effectively kill the target cellswhile sparing the ‘off-target’ cells will be assessed by an in-vivo CTLassay.

In some embodiments, transduced T-cells with iCAR or aCAR or both iCARand aCAR will be injected i.v. to naïve NOD/SCID/γc- or similar mice andup to several hours later, target cells expressing iCAR, aCAR or bothwill be injected. In some embodiments, these targets will be labeledwith either CFSE/CPDE or similar cell trace dye in differentconcentrations (high, medium and low) which will allow furtherdiscrimination between them. In some embodiments, percentage of specifickilling will be calculated, as described in Example 5.

ii. Tumor Growth Kinetics in Human Xenograft Mouse Models

In some embodiments, the tumor cells express either the iCAR target,aCAR target or both. In some embodiments, an aCAR tumor cell line couldbe the CD19 positive NALM 6 (ATCC, human BALL cell line). In someembodiments, tumor cells that express both the aCAR and iCAR (i.e.‘off-tumor’ cells) are NALM 6 engineered to express the iCAR epitope(for example HLA-A2) thereby representing the healthy cells. In someembodiments, NALM 6 and NA1M 6-HLA-A2 can also be engineered to expressa reporter gene (e.g. firefly luciferase), for easy detection.

In some embodiments, monitoring will be conducted by measuring tumorvolume by mechanical means (caliper) and also by using in-vivo imagingsystems (IVIS). In some embodiments, tumor burden can be quantified, andinfiltrating T-cell populations can be analyzed by FACS.

iii. Toxicity and Tumor Growth Kinetics in Transgenic Mouse Models

In some embodiments, transgenic mice that express the human aCAR andiCAR targets will also be used to determine the efficacy of thetransduced T-cells. In some embodiments, system will allow us to monitorefficacy and toxicity issues.

C. In Vivo Uses: Treatment, Biomarkers

In yet another aspect, the present invention provides a method ofselecting a personalized biomarker for a subject having a tumorcharacterized by LOH, the method comprising (i) obtaining a tumor biopsyfrom the subject; (ii) obtaining a sample of normal tissue from thesubject, e.g., PBMCs; and (iii) identifying a single allelic variant ofa polymorphic cell surface epitope that is not expressed by cells of thetumor due to LOH, but that is expressed by the cells of the normaltissue, thereby identifying a personalized biomarker for the subject.

In some embodiments, the biomarker is used to customize a treatment ofthe subject, so the method further comprises the steps of treatingcancer in a patient having a tumor characterized by LOH, comprisingadministering to the patient an effector immune cell as defined above,wherein the iCAR is directed to the single allelic variant identified in(iii). In some embodiments, the present invention provides a method ofselecting a personalized biomarker for a subject having a tumorcharacterized by LOH, the method comprising (i) obtaining a tumor biopsyfrom the subject; (ii) obtaining a sample of normal tissue from thesubject, e.g. PBMCs; (iii) identifying a single allelic variant of apolymorphic cell surface epitope that is not expressed by cells of thetumor due to LOH, but that is expressed by the cells of the normaltissue, based on the LOH candidate score, wherein an allelic variant isidentified as a personalized biomarker for the subject.

In a further aspect, the present invention provides a method fortreating cancer in a patient having a tumor characterized by LOH,comprising administering to the patient an effector immune cell asdefined above, wherein the iCAR is directed to a single allelic variantencoding a polymorphic cell surface epitope absent from cells of thetumor due to loss of heterozygosity (LOH) but present at least on allcells of related mammalian normal tissue of the patient.

In a similar aspect, the present invention provides a method of reducingtumor burden in a subject having a tumor characterized by LOH,comprising administering to the patient an effector immune cell asdefined above, wherein the iCAR is directed to a single allelic variantencoding a polymorphic cell surface epitope absent from cells of thetumor due to loss of heterozygosity (LOH) but present at least on allcells of related mammalian normal tissue of the patient or at least onvital tissues the aCAR is expressed in.

In another similar aspect, the present invention provides a method ofincreasing survival of a subject having a tumor characterized by LOH,comprising administering to the patient an effector immune cell asdefined above, wherein the iCAR is directed to a single allelic variantencoding a polymorphic cell surface epitope absent from cells of thetumor due to loss of heterozygosity (LOH) but present at least on allcells of related mammalian normal tissue of the patient.

In still a further aspect, the present invention is directed to a safeeffector immune cell as defined above for use in treating, reducingtumor burden in, or increasing survival of, a patient having a tumorcharacterized by LOH, wherein the iCAR is directed to a single allelicvariant encoding a polymorphic cell surface epitope absent from cells ofthe tumor due to loss of heterozygosity (LOH) but present at least onall cells of related mammalian normal tissue of the patient.

In yet a further aspect, the present invention is directed to a methodfor treating cancer in a patient having a tumor characterized by LOHcomprising: (i) identifying or receiving information identifying asingle allelic variant of a polymorphic cell surface epitope that is notexpressed by cells of the tumor due to LOH, but that is expressed by thecells of the normal tissue, (ii) identifying or receiving informationidentifying a non-polymorphic cell surface epitope of an antigen or asingle allelic variant of a polymorphic cell surface epitope, whereinsaid epitope is a tumor-associated antigen or is shared by cells atleast of related tumor and normal tissue in said cancer patient; (iii)selecting or receiving at least one nucleic acid molecule defining aniCAR as defined herein above and at least one nucleic acid moleculecomprising a nucleotide sequence encoding an aCAR as defined hereinabove, or at least one vector as defined herein above, wherein the iCARcomprises an extracellular domain that specifically binds to a cellsurface epitope of (i) and the aCAR comprises an extracellular domainthat specifically binds to a cell surface epitope of (ii); (iv)preparing or receiving at least one population of safe redirectedeffector immune cells by transfecting effector immune cells with thenucleic acid molecules of (iii) or transducing effector immune cellswith the vectors of (iii); and (v) administering to said cancer patientat least one population of safe redirected immune effector cells of(iv).

In a similar aspect, the present invention provides at least onepopulation of safe redirected immune effector cells for treating cancerin a patient having a tumor characterized by LOH, wherein the saferedirected immune cells are obtained by (i) identifying or receivinginformation identifying a single allelic variant of a polymorphic cellsurface epitope that is not expressed by cells of the tumor due to LOH,but that is expressed by the cells of the normal tissue, (ii)identifying or receiving information identifying a non-polymorphic cellsurface epitope of an antigen or a single allelic variant of apolymorphic cell surface epitope, wherein said epitope is atumor-associated antigen or is shared by cells at least of related tumorand normal tissue in said cancer patient; (iii) selecting or receivingat least one nucleic acid molecule defining an iCAR as defined hereinabove and at least one nucleic acid molecule comprising a nucleotidesequence encoding an aCAR as defined herein above, or at least onevector as defined herein above, wherein the iCAR comprises anextracellular domain that specifically binds to a cell surface epitopeof (i) and the aCAR comprises an extracellular domain that specificallybinds to a cell surface epitope of (ii); (iv) preparing or receiving atleast one population of safe redirected effector immune cells bytransfecting effector immune cells with the nucleic acid molecules of(iii) or transducing effector immune cells with the vectors of (iii).

In some embodiments referring to any one of the above embodimentsdirected to treatment of cancer or safe immune effector cells for use intreatment of cancer, (i) the extracellular domain of the iCARspecifically binds a single allelic variant of a polymorphic cellsurface epitope of an antigen, which is a different antigen than that towhich the extracellular domain of the aCAR binds; (ii) the extracellulardomain of said iCAR specifically binds a single allelic variant of adifferent polymorphic cell surface epitope of the same antigen to whichthe extracellular domain of said aCAR binds; or (iii) the extracellulardomain of said iCAR specifically binds a different single allelicvariant of the same polymorphic cell surface epitope to which theextracellular domain of said aCAR binds.

In some embodiments, the treating results in reduced on-target,off-tumor reactivity, as compared with a treatment comprisingadministering to the cancer patient at least one population of immuneeffector cells expressing an aCAR of (iii) but lacking and iCAR of(iii).

In some embodiments, the safe effector immune cells used for treatingcancer as defined above express on their surface an aCAR comprising anextracellular domain that specifically binds to a tumor-associatedantigen or a non-polymorphic cell surface epitope of an antigen and aniCAR comprising an extracellular domain that specifically binds a singleallelic variant of a polymorphic cell surface epitope of an antigenexpressed at least in a tissue of origin of the tumor or of ahousekeeping protein, which is a different antigen than that to whichthe extracellular domain of said aCAR binds.

In some embodiments, the safe effector immune cells used for treatingcancer as defined above express on their surface an aCAR comprising anextracellular domain that specifically binds to a tumor-associatedantigen or a non-polymorphic cell surface epitope of an antigen and aniCAR comprising an extracellular domain that specifically binds a singleallelic variant of a polymorphic cell surface epitope of an antigenexpressed at least in a tissue of origin of the tumor or of ahousekeeping protein, such as an HLA genes (including for example,HLA-A, HLA-B, HLA-C, HLA-G, HLA-E, HLA-F, HLA-K, HLA-L, HLA-DM, HLA-DO,HLA-DP, HLA-DQ, or HLA-DR) which is a different antigen than that towhich the extracellular domain of said aCAR binds.

In some embodiments, the safe effector immune cells used for treatingcancer as defined above express on their surface an aCAR comprising anextracellular domain that specifically binds to a tumor-associatedantigen or a non-polymorphic cell surface epitope of an antigen and aniCAR comprising an extracellular domain that specifically binds a singleallelic variant of a polymorphic cell surface epitope of an antigenexpressed at least in a tissue of origin of the tumor, such as an HLA-A,which is a different antigen than that to which the extracellular domainof said aCAR binds.

In some embodiments, more than one population of immune effector cellsare administered, and the different populations express different pairsof aCARs and iCARs having specific binding to cell surface epitopes ofdifferent gene products.

In some embodiments, the safe effector immune cells used in the methodof treating cancer are selected from T cells, natural killer cells orcytokine-induced killer cells. In some embodiments, the safe effectorimmune cell is autologous or universal (allogeneic) effector cells. Insome embodiments, the iCAR used in any one of the methods of treatingcancer defined above is directed to all tissues of the patient on whichthe target-antigen of the aCAR is present, wherein the target antigen ofthe aCAR is a non-polymorphic cell surface epitope of an antigen or asingle allelic variant of a polymorphic cell surface epitope is present,and said epitope is a tumor-associated antigen or is shared at least bycells of related tumor and normal tissue.

In some embodiments, the cancer is selected from Acute Myeloid Leukemia[LAML], Adrenocortical carcinoma [ACC], Bladder Urothelial Carcinoma[BLCA], Brain Lower Grade Glioma [LGG], Breast invasive carcinoma[BRCA], Cervical squamous cell carcinoma and endocervical adenocarcinoma[CESC], Cholangiocarcinoma [CHOL], Colon adenocarcinoma [COAD],Esophageal carcinoma [ESCA], Glioblastoma multiforme [GBM], Head andNeck squamous cell carcinoma [HNSC], Kidney Chromophobe [KICH], Kidneyrenal clear cell carcinoma [KIRC], Kidney renal papillary cell carcinoma[KIRP], Liver hepatocellular carcinoma [LIHC], Lung adenocarcinoma[LUAD], Lung squamous cell carcinoma [LUSC], Lymphoid Neoplasm DiffuseLarge B-cell Lymphoma [DLBC], Mesothelioma [MESO], Ovarian serouscystadenocarcinoma [OV], Pancreatic adenocarcinoma [PARD],Pheochromocytoma and Paraganglioma [PCPG], Prostate adenocarcinoma[PRAD], Rectum adenocarcinoma [READ], Sarcoma [SARC], Skin CutaneousMelanoma [SKCM], Stomach adenocarcinoma [STAD], Testicular Germ CellTumors [TGCT], Thymoma [THYM], Thyroid carcinoma [THCA], UterineCarcinosarcoma [UCS], Uterine Corpus Endometrial Carcinoma [UCEC], UvealMelanoma [UVM].

In some embodiments, the iCAR and/or pCAR for use in the treatment ofcancer is any iCAR and/or pCAR described herein. In some embodiments,the iCAR and/or pCAR used to treat the cancer, such as any one of thecancer types recited above, is directed against or specifically binds toa single allelic variant of an HLA genes (including for example, HLA-A,HLA-B, HLA-C, HLA-G, HLA-E, HLA-F, HLA-K, HLA-L, HLA-DM, HLA-DO, HLA-DP,HLA-DQ, or HLA-DR, HLA-B gene or HLA-C gene or against a single allelicvariant of a gene listed Table 8 In some embodiments, the iCAR used totreat the cancer, such as any one of the cancer types recited above, isdirected against or specifically binds to a single allelic variant of anHLA-A gene, HLA-B gene or HLA-C gene or against a single allelic variantof a gene listed Table 8; and the aCAR used to treat the cancer, such asany one of the cancer types recited above, is directed against orspecifically binds to, a non-polymorphic cell surface epitope selectedfrom the antigens listed in Table 1, such as CD19.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The compositions may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multidose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen free water, before use.

The compositions may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

For administration by inhalation, the compositions for use according tothe present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin, for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

For purposes of clarity, and in no way limiting the scope of theteachings, unless otherwise indicated, all numbers expressingquantities, percentages or proportions, and other numerical valuesrecited herein, should be interpreted as being preceded in all instancesby the term “about.” Accordingly, the numerical parameters recited inthe present specification are approximations that may vary depending onthe desired outcome. For example, each numerical parameter may beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

The term “about” as used herein means that values of 10% or less aboveor below the indicated values are also included.

Exemplary Embodiments

In some embodiments, the methods of the present invention provide forthe following exemplary embodiments.

-   1. A nucleic acid molecule comprising a nucleotide sequence encoding    an inhibitory chimeric antigen receptor (iCAR) or protective    chimeric antigen receptor (pCAR) capable of preventing or    attenuating undesired activation of an effector immune cell, wherein    the iCAR or pCAR comprises an extracellular domain that specifically    binds to a single allelic variant of a polymorphic cell surface    epitope absent from mammalian tumor cells due to loss of    heterozygosity (LOH) but present at least on all cells of related    mammalian normal tissue; and an intracellular domain comprising at    least one signal transduction element that inhibits an effector    immune cell.-   2. The nucleic acid molecule of claim 1, wherein the polymorphic    cell surface epitope is of a housekeeping gene product, such as an    HLA gene, a G-protein-coupled receptor (GPCR), an ion channel or a    receptor tyrosine kinase, preferably an HLA-A, HLA-B or HLA-C; or a    polymorphic cell surface epitope of a gene selected from Table 8.-   3. The nucleic acid molecule claim 1, wherein said extracellular    domain comprises (i) an antibody, derivative or fragment thereof,    such as a humanized antibody; a human antibody; a functional    fragment of an antibody; a single-domain antibody, such as a    Nanobody; a recombinant antibody; and a single chain variable    fragment (ScFv); (ii) an antibody mimetic, such as an affibody    molecule; an affilin; an affimer; an affitin; an alphabody; an    anticalin; an avimer; a DARPin; a fynomer; a Kunitz domain peptide;    and a monobody; or (iii) an aptamer.-   4. The nucleic acid molecule of claim 1, wherein said mammalian    tissue is human tissue and said related mammalian normal tissue is    normal tissue from which the tumor developed.-   5. The nucleic acid molecule of claim 1, wherein said effector    immune cell is a T cell, a natural killer cell or a cytokine-induced    killer cell.-   6. The nucleic acid molecule of claim 1, wherein said at least one    signal transduction element capable of inhibiting an effector immune    cell is homologous to a signal transduction element of an immune    checkpoint protein.-   7. The nucleic acid molecule of claim 6, wherein said immune    checkpoint protein is selected from the group consisting of PD1;    CTLA4; BTLA; 2B4; CD160; CEACAM, such as CEACAM1; KIRs, such as    KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1,    KIR3DL2, KIR3DL3, LIR1, LIR2, LIR3, LIR5, LIR8 and CD94-NKG2A; LAG3;    TIM3; V-domain Ig suppressor of T cell activation (VISTA);    STimulator of INterferon Genes (STING); immunoreceptor    tyrosine-based inhibitory motif (ITIM)-containing proteins, T cell    immunoglobulin and ITIM domain (TIGIT), and adenosine receptor (e.g.    A2aR).-   8. The nucleic acid molecule of claim 1, wherein said extracellular    domain is fused through a flexible hinge and transmembrane canonic    motif to said intracellular domain.-   9. A vector comprising a nucleic acid molecule of any one of claims    1 to 8 and at least one control element, such as a promoter,    operably linked to the nucleic acid molecule.-   10. The vector of claim 9, further comprising a nucleic acid    molecule comprising a nucleotide sequence encoding an aCAR    comprising an extracellular domain specifically binding a    non-polymorphic cell surface epitope of an antigen or a single    allelic variant of a polymorphic cell surface epitope, wherein said    epitope is a tumor-associated antigen or is shared at least by cells    of related tumor and normal tissue, and an intracellular domain    comprising at least one signal transduction element that activates    and/or co-stimulates an effector immune cell.-   11. The vector of claim 10, wherein the extracellular domain of the    aCAR specifically binds to a non-polymorphic cell surface epitope of    an antigen and the extracellular domain of the iCAR specifically    binds a single allelic variant of a polymorphic cell surface epitope    of a different antigen than that to which the extracellular domain    of said aCAR binds.-   12. The vector of claim 10 or 11, wherein the extracellular domain    of the aCAR specifically binds to a non-polymorphic cell surface    epitope selected from the antigens listed in Table 1, such as CD19.-   13. The vector of claim 10, wherein said at least one signal    transduction element that activates or co-stimulates an effector    immune cell is homologous to an immunoreceptor tyrosine-based    activation motif (ITAM) of for example CD3ζ or FcRγ chains; an    activating killer cell immunoglobulin-like receptor (KIR), such as    KIR2DS and KIR3DS, or an adaptor molecule such as DAP12; or a    co-stimulatory signal transduction element of for example CD27,    CD28, ICOS, CD137 (4-1BB) or CD134 (OX40).-   14. The vector of claim 10, wherein the nucleotide sequence    comprises an internal ribosome entry site (IRES) between the    nucleotide sequence encoding for the aCAR and the nucleotide    sequence encoding for the iCAR-   15. The vector of claim 14, wherein the nucleotide sequence encoding    for the aCAR is downstream of the nucleotide sequence encoding for    the iCAR.-   16. The vector of claim 10, wherein the nucleotide sequence    comprises a viral self-cleaving 2A peptide between the nucleotide    sequence encoding for the aCAR and the nucleotide sequence encoding    for the iCAR.-   17. The vector of claim 16, wherein the viral self-cleaving 2A    peptide is selected from the group consisting of T2A from Thosea    asigna virus (TaV), F2A from Foot-and-mouth disease virus (FMDV),    E2A from Equine rhinitis A virus (ERAV) and P2A from Porcine    teschovirus-1 (PTV1).-   18. The vector of claim 10, comprising a nucleotide sequence    encoding said constitutive aCAR linked via a flexible linker to said    iCAR.-   19. A method of preparing an inhibitory chimeric antigen receptor    (iCAR) capable of preventing or attenuating undesired activation of    an effector immune cell, as defined in claims 1 to 8, the method    comprising:    -   (i) retrieving a list of human genomic variants of        protein-encoding genes from at least one database of known        variants;    -   (ii) filtering the list of variants retrieved in (i) by:        -   (a) selecting variants resulting in an amino acid sequence            variation in the protein encoded by the respective gene as            compared with its corresponding reference allele,        -   (b) selecting variants of genes wherein the amino acid            sequence variation is in an extracellular domain of the            encoded protein,        -   (c) selecting variants of genes that undergo loss of            heterozygosity (LOH) at least in one tumor, and        -   (d) selecting variants of genes that are expressed at least            in a tissue of origin of the at least one tumor in which            they undergo LOH according to (c), thereby obtaining a list            of variants having an amino acid sequence variation in an            extracellular domain in the protein encoded by the            respective gene lost in the at least one tumor due to LOH            and expressed at least in a tissue of origin of the at least            one tumor;    -   (iii) defining a sequence region comprising at least one single        variant from the list obtained in (ii), sub-cloning and        expressing the sequence region comprising the at least one        single variant and a sequence region comprising the        corresponding reference allele thereby obtaining the respective        epitope peptides;    -   (iv) selecting an iCAR binding domain, which specifically binds        either to the epitope peptide encoded by the cloned sequence        region, or to the epitope peptide encoded by the corresponding        reference allele, obtained in (iii); and    -   (vii) preparing iCARs as defined in any one of claims 1 to 8,        each comprising an iCAR binding domain as defined in (iv).-   20. The method of claim 19, wherein the minor allele frequency for    each variant equals or exceeds 1, 2, 3, 4 or 5%.-   21. A method for preparing a safe effector immune cell    comprising: (i) transfecting a TCR-engineered effector immune cell    directed to a tumor-associated antigen with a nucleic acid molecule    comprising a nucleotide sequence encoding an iCAR of any one of    claims 1 to 8 or transducing the cells with a vector of claim 9;    or (ii) transfecting a naïve effector immune cell with a nucleic    acid molecule comprising a nucleotide sequence encoding an iCAR of    any one of claims 1 to 8 and a nucleic acid molecule comprising a    nucleotide sequence encoding an aCAR defined in any one of claims 10    to 13; or transducing an effector immune cell with a vector of any    one of claims 10 to 18.-   22. A safe effector immune cell obtained by the method of claim 21.-   23. The safe effector immune cell of claim 22, expressing on its    surface an aCAR comprising an extracellular domain that specifically    binds to a non-polymorphic cell surface epitope of an antigen and an    iCAR comprising an extracellular domain that specifically binds a    single allelic variant of a polymorphic cell surface epitope of a    different antigen to which the extracellular domain of said aCAR    binds.-   24. The safe effector immune cell of claim 22 or 23, wherein the    extracellular domain of the aCAR specifically binds to a    non-polymorphic cell surface epitope selected from the antigens    listed in Table 1, such as CD19.-   25. The safe effector immune cell of claim 22, wherein the aCAR and    the iCAR are present on the cell surface as separate proteins.-   26. The safe effector immune cell of claim 22, wherein the    expression level of said nucleotide sequence encoding the iCAR is    greater than or equal to the expression level of the nucleotide    sequence encoding the aCAR.-   27. A method of selecting a personalized biomarker for a subject    having a tumor characterized by LOH, the method comprising    -   (i) obtaining a tumor biopsy from the subject;    -   (ii) obtaining a sample of normal tissue from the subject, e.g.        PBMCs;    -   (iii) identifying a single allelic variant of a polymorphic cell        surface epitope that is not expressed by cells of the tumor due        to LOH, but that is expressed by the cells of the normal tissue,    -   thereby identifying a personalized biomarker for the subject.-   28. A method for treating cancer in a patient having a tumor    characterized by LOH, comprising administering to the patient an    effector immune cell of claim 22, wherein the iCAR is directed to a    single allelic variant encoding a polymorphic cell surface epitope    absent from cells of the tumor due to loss of heterozygosity (LOH)    but present at least on all cells of related mammalian normal tissue    of the patient.-   29. A safe effector immune cell of claim 22 for use in treating    patient having a tumor characterized by LOH, wherein the iCAR is    directed to a single allelic variant encoding a polymorphic cell    surface epitope absent from cells of the tumor due to loss of    heterozygosity (LOH) but present at least on all cells of related    mammalian normal tissue of the patient.-   30. The safe effector immune cell for the use of claim 29, wherein    the treating results in reduced on-target, off-tumor reactivity, as    compared with a treatment comprising administering to the cancer    patient at least one population of immune effector cells expressing    an aCAR of (iii) but lacking and iCAR of (iii).-   31. The safe effector immune cell for the use of claim 29,    expressing on its surface an aCAR comprising an extracellular domain    that specifically binds to a tumor-associated antigen or a    non-polymorphic cell surface epitope of an antigen and an iCAR    comprising an extracellular domain that specifically binds a single    allelic variant of a polymorphic cell surface epitope of an antigen    expressed at least in a tissue of origin of the tumor or of a    housekeeping protein, such as an HLA-A, which is a different antigen    than that to which the extracellular domain of said aCAR binds.-   32. The safe effector immune cell for the use of claim 28, which is    an autologous or a universal (allogeneic) effector cell.-   33. The safe effector immune cell for the use of any one of claims    28 to 32, selected from a T cell, natural killer cell or    cytokine-induced killer cell.-   34. A combination of two or more nucleic acid molecules, each one    comprising a nucleotide sequence encoding a different member of a    controlled effector immune cell activating system, said nucleic acid    molecules forming a single continues nucleic acid molecule or    comprising two or more separate nucleic acid molecules, wherein the    controlled effector immune activating system directs effector immune    cells to kill tumor cells that have lost one or more chromosomes or    fractions thereof due to Loss of Heterozygosity (LOH) and spares    cells of related normal tissue, and wherein    -   (a) the first member comprises an activating chimeric antigen        receptor (aCAR) polypeptide comprising a first extracellular        domain that specifically binds to a non-polymorphic cell surface        epitope of an antigen or to a single allelic variant of a        different polymorphic cell surface epitope and said        non-polymorphic or polymorphic cell surface epitope is a        tumor-associated antigen or is shared by cells of related        abnormal and normal mammalian tissue; and    -   (b) the second member comprises a regulatory polypeptide        comprising a second extracellular domain that specifically binds        to a single allelic variant of a polymorphic cell surface        epitope not expressed by an abnormal mammalian tissue due to LOH        but present on all cells of related mammalian normal tissue.-   35. The combination of claim 34, wherein the first member is    selected from:    -   (a) a constitutive aCAR further comprising an intracellular        domain comprising at least one signal transduction element that        activates and/or co-stimulates an effector immune cell; and    -   (b) a conditional aCAR further comprising an intracellular        domain comprising a first member of a binding site for a        heterodimerizing small molecule and optionally at least one        co-stimulatory signal transduction element, but lacking an        activating signal transduction element; and the second member        is:    -   (c) an inhibiting chimeric antigen receptor (iCAR) further        comprising an intracellular domain comprising at least one        signal transduction element that inhibits an effector immune        cell; or    -   (d) a protective chimeric antigen receptor (pCAR) further        comprising an extracellular regulatory region comprising a        substrate for a sheddase; a transmembrane canonic motif        comprising a substrate for an intramembrane-cleaving protease;        and an intracellular domain, said intracellular domain        comprising at least one signal transduction element that        activates and/or co-stimulates an effector immune cell and a        second member of a binding site for a heterodimerizing small        molecule.-   36. The combination of claim 34 or 35, wherein:    -   (i) the extracellular domain of the iCAR or pCAR specifically        binds a single allelic variant of a polymorphic cell surface        epitope of an antigen, which is a different antigen than that to        which the extracellular domain of the aCAR binds    -   (ii) the extracellular domain of said pCAR or iCAR specifically        binds a single allelic variant of a different polymorphic cell        surface epitope of the same antigen to which the extracellular        domain of said aCAR binds; or    -   (iii) the extracellular domain of said pCAR or iCAR specifically        binds a different single allelic variant of the same polymorphic        cell surface epitope to which the extracellular domain of said        aCAR binds.-   37. The combination of claim 34, wherein said substrate for a    sheddase is a substrate for a disintegrin and metalloproteinase    (ADAM) or a beta-secretase 1 (BACE1).-   38. The combination of claim 37, wherein said substrate forms part    of the extracellular domain and comprises Lin 12/Notch repeats and    an ADAM protease cleavage site.-   39. The combination of claim 34, wherein said substrate for an    intramembrane-cleaving protease is a substrate for an SP2, a    y-secretase, a signal peptide peptidase (spp), a spp-like protease    or a rhomboid protease.-   40. The combination of claim 39, wherein said substrate forms part    of the transmembrane canonic motif and is homologous to/derived from    a transmembrane domain of Notch, ErbB4, E-cadherin, N-cadherin,    ephrin-B2, amyloid precursor protein or CD44.-   41. The combination of claim 34, comprising a nucleotide sequence    encoding an extracellular domain and an intracellular domain of said    conditional aCAR as separate proteins, wherein each domain is    independently fused to a transmembrane canonic motif and comprises a    different member of a binding site for a heterodimerizing small    molecule.-   42. The combination of claim 34, wherein each one of said first and    second member of said binding site for a heterodimerizing small    molecule is derived from a protein selected from:    -   (i) Tacrolimus (FK506) binding protein (FKBP) and FKBP;    -   (ii) FKBP and calcineurin catalytic subunit A (CnA);    -   (iii) FKBP and cyclophilin;    -   (iv) FKBP and FKBP-rapamycin associated protein (FRB);    -   (v) gyrase B (GyrB) and GyrB;    -   (vi) dihydrofolate reductase (DHFR) and DHFR;    -   (vii) DmrB homodimerization domain (DmrB) and DmrB;    -   (viii) a PYL protein (a.k.a. abscisic acid receptor and as RCAR)        and ABI;    -   (ix) GAI Arabidopsis thaliana protein (a.k.a Gibberellic Acid        Insensitive and DELLA protein GAL GAI) and GID1 Arabidopsis        thaliana protein (also known as Gibberellin receptor GID1;        GID1).

Lengthy Tables

The patent application contains a lengthy table section. Copies of thetables are submitted concurrently herewith on CD-ROM.

EXAMPLES

With regard to the examples, the following terminology is employed.

When the term chromosome is employed, this generally refers to thechromosome the SNP lies on. For the SNP analysis, position refers to thegenomic position of the SNP (assembly GRCh37.p13). The snp id when usedrefers to the dbSNP rs ID, where one exists.

The term “ref” refers to the reference nucleotide allele. The term “alt”refers to the alternative nucleotide allele.

The term “quality” refers to the quality score from Exome AggregationConsortium (ExAC). The term “filter status” refers to filter informationfrom ExAC.

The term “allele_frequency” refers to the global allele frequency fromExAC. The term “max_allele_frequency” refers to the global allelefrequency of most common alternative allele (generally, this is onlyrelevant when the SNP has more than two alternative alleles at the samesite, and this can often mean sequencing errors anyway).

The term “het_allele_count” refers to the number of participants in ExACwho were heterozygotes. The term “AFR_AF” refers to minor allelefrequency from African genomes. The term “AMR_AF” refers to minor allelefrequency in Latino genomes. The term “EAS_AF” refers to minor allelefrequency in East Asian genomes. The term “FIN_AF” refers to minorallele frequency in Finnish genomes. The term “NFE_AF” refers to minorallele frequency in Non-Finnish-European genomes. The term “OTH_AF”refers to minor allele frequency in Other genomes. The term “SAS_AF”refers to minor allele frequency in South Asian genomes.

The term “max_AF” refers to maximum minor allele frequency amongst thepopulations categorized in ExAC (0.5 is maximum allowable allelefrequency).

The term “gene” refers to the HUGO symbol of the gene in which the SNPfalls.

The term “hgnc_ID” refers to the HUGO Gene Nomenclature Committeenumeric ID of the gene in which the SNP falls.

The term “consequence” refers to the impact of the SNP on the translatedprotein product. Can be one of several, including: missense_variant,frameshift_variant, inframe_deletion, stop_gained.

The term “protein_consequence” reports the amino acid substitution andthe location thereof on the reference protein transcript (e.g.p.Arg482G1n).

The term “aa_affected” refers to the numeric location of the affectedamino acid on the consensus protein transcript.

The term “allele_1” refers to the amino acid encoded by the referenceallele.

The term “allele_2” refers to the amino acid encoded by the alternativeallele.

The term “sift_score” refers to the score and interpretation of thepredicted functional effect of the amino acid substitution by the SIFTalgorithm. Uses version sift5.2.2. Scores range from 0-1. A low scoremeans than an amino acid substitution is more likely to be tolerated.

The term “polyphen_score” refers to the score and interpretation of thepredicted functional effect of the amino acid substitution by thepolyphen algorithm. Uses PolyPhen (v2.2.2). Scores range from 0-1. A lowscore means than an amino acid substitution is more likely to bedeleterious.

The term “polyphen_numeric” refers to the extracted numeric only scorefrom the polyphen algorithm.

The term “protein_domains_affected” refers to the predicted proteindomains based on the following algorithms: Gene3D, hmmpanther, Prosite.

The term “BLOSUM_score” refers to the score for the amino acidsubstitution based on the BLOSUM62 matrix fromhttps://www.ncbi.nlm.nih.gov/IEB/ToolBox/C_DOC/lxr/source/data/BLOSUM62.A negative score indicates an amino acid substitution that has occurredless frequently over time in evolution (more likely to affect proteinfunction).

The term “allele_1_one_letter” refers to the one letter amino acid codeof the reference amino acid allele.

The term “allele_2_one_letter” refers to the one letter amino acid codeof the alternative amino acid allele.

The term “mono_allelic_expression” refers to whether or not the genethat the SNP falls in undergoes mono-allelic expression in humans. Thedatabase established by Savova et al. was used for this annotation⁷. A 1in this column indicates that the gene displays mono-allelic expression.A 0 in this column indicates that the gene did not display mono-allelicexpression in the Savova et al. database. An NA in this column meansthat the gene was not annotated in the Savova et al. paper.

The term “extracellular” refers to whether or not the SNP falls in anextracellular domain of the affected protein. A 1 in this columnindicates that the SNP is in an extracellular domain and a 0 indicatesthat it is not. Uniprot was used for annotation of protein domains.

The term “Pdb_id” refers to the protein databank ID of the affectedprotein if it exists. In the case where many protein databank entriesexist for one protein, only the first ID is included.

The term “aa_context_21aa_allele_1” refers to A 21 amino acid windowsurrounding the SNP amino acid on the consensus protein sequence. Thesequence consists of the 10 amino acids from the preceding part of theconsensus protein sequence. A check was made to ensure that thereference amino acid matched the consensus protein sequence at theaffected position. If these two amino acids were not the same, then theentry reads “discrepancy with uniprot fasta based on consensus isoform”.

The term “aa_context_21aa_allele_2: The same amino acid window as above,but inserting amino acid allele 2 into the middle.

The term “gtex_mean: Average gene expression across tissues (in RPKM).This consists of the mean value of the median RPKM values across tissuesfrom GTEX. For example, if the values for a given gene were Lung(median)=3, Breast (median)=2, Pancreas (median)=5, then the valuereported in this entry would be 3.33.

The term “gtex_min: The lowest gene expression for a tissue across alltissues. This value is derived from the list of the median values ofgene expression across all tissues. For example, if the values for agiven gene were Lung (median)=3, Breast (median)=2, Pancreas (median)=5,then the value reported in this entry would be 2.

The term “gtex_max: The highest gene expression for a tissue across alltissues. This value is derived from the list of the median values ofgene expression across all tissues. For example, if the values for agiven gene were Lung (median)=3, Breast (median)=2, Pancreas (median)=5,then the value reported in this entry would be 5.

The term “gtex_std_dev: The standard deviation of gene expression valuesacross tissues for a given gene. For example, if the values for a givengene were Lung (median)=3, Breast (median)=2, Pancreas (median)=5, thenthe value reported in this entry would be 1.5.

The term “cell_surface_protein_atlas: A binary marker for whether or notthe protein was annotated as a membrane protein in the cell surfaceprotein atlas (wlab.ethz.ch/cspa/). A 1 indicates that the gene wasannotated as a membrane protein in this database.

The term “human_protein_atlas_membrane_proteins: A binary marker forwhether or not the protein was annotated as a membrane protein in thehuman protein atlas (https://www.proteinatlas.org/). A 1 indicates thatthe gene was annotated as a membrane protein in this database.

The term “subcellular_map_proteome_membrane_proteins: A binary markerfor whether or not the protein was annotated as a membrane protein inthe subcellular map of the proteome(http://science.sciencemag.org/content/early/2017/05/10/science.aa13321/).A 1 indicates that the gene was annotated as a membrane protein in thisdatabase.

The term “n_membrane_databases_w_gene: The total number of databaseswith the gene annotated as a gene that is expressed on the cellmembrane. Maximum=3, minimum=0.

The term “membrane_protein_call: A textual interpretation of the numberof membrane databases that the included the gene. If the gene wasincluded in one database, then the call is a “low-confidence” membraneprotein. If the gene was included in two databases, then the call is a“medium-confidence” membrane protein. If the gene was included in threedatabases, then the call is a “high-confidence” membrane protein.

The term “ratio_gtex_std_dev_to_mean: The ratio of the standarddeviation of gene expression across tissues over the mean geneexpression across tissues. For example, if the values for a given genewere Lung (median)=3, Breast (median)=2, Pancreas (median)=5, then thevalue reported in this entry would be 1.5/3.33=0.45. This is meant to bea measure of the uniformity of expression across tissues. A low valueindicates that the gene is uniformly expressed. A high value suggeststhat the gene tends to be expressed in some tissues and not others.

The term “universally_expressed: A binary marker of whether a gene seemsto be universally expressed. A gene is said to be universally expressedif the gtex_mean is >10, the gtex_min. The term “>1, andratio_gtex_std_dev_to_mean <1. A 1 in this column indicates that thegene in question met these criteria.

The term “disease: the TCGA barcode for the disease analyzed for LOHdata in this row of the spreadsheet.

The term “mean_expression_in_tissue: The mean gene expression in thetissue analyzed. Several tissue categorizations may map onto a singleTCGA tumor type. The mapping from tissues in GTEX to TCGA tumor types isgiven in the file “tcga_disease_tissue_lookup.txt”. A representativesample is given below:

tcga_disease gtex_tissues acc Adrenal.Gland blca Bladder brca Breast . .. Mammary.Tissue cesc Cervix . . . Endocervix, Cervix . . . Ectocervix

The term “mean_expression_in_other_tissues: The mean gene expression inall other tissues except for the tissue analyzed. For example, if thegene being analyzed was PSMA (a prostate specific gene), then this valuewould be very low when the tumor type analyzed was PRAD (prostateadenocarcinoma).

The term “cohens_d: The Cohen's d measure of the separation of theexpression in the tissue analyzed vs all other tissues. This is meant tobe a measure of how much this gene is uniquely expressed in the tissueanalyzed. A high Cohen's d would suggest that this gene is uniquelyexpressed in the tissue analyzed and therefore might be a good aCARtarget.

The term “proportion_w_LOH_relative: The proportion of tumors in thetumor type analyzed that display evidence of LOH. The threshold forcalling a genomic segment suggesting LOH was −0.1 (in relative copynumber units). The relative copy number of a segment was the log of thecopy number signal in the tumor divided by the copy number signal in thematched normal. These data were obtained from the cbio portal and thetechnique was validated in part 1.

The term “CI_95_low_relative: The lower boundary of the 95% confidenceinterval on the proportion of tumors undergoing LOH at this locus. Theprop.test function in R was used for this calculation. This functioncalculates a binomial confidence interval with Yates' continuitycorrection.

The term “CI_95_high_relative: The upper boundary of the 95% confidenceinterval on the proportion of tumors undergoing LOH at this locus. Theprop.test function in R was used for this calculation. This functioncalculates a binomial confidence interval with Yates' continuitycorrection.

The term “mutsig_hits_on_chr: The genes on the same chromosome as theSNP that pass statistical significance (q-value <0.25) for being driversin cancer. The Mutsig 2.0 algorithm was used. The format is “Genesymbol, q=q-value; Gene symbol 2, . . . ”

The term “tsg_on_chr_mutated_in_disease: A binary indicator variable forwhether or not one of the genes passing statistically significance frommutsig is a tumor suppressor gene. The list of tumor suppressor genesused for this annotation was the list from the table published byVogelstein et al⁹. A 1 in this column indicates that the gene isannotated as a tumor suppressor gene.

The term “hallmark_tsg_on_chr_mutated_in_disease: A binary indicatorvariable for whether any of the genes identified as significantlymutated in the tumor type analyzed and on the same chromosome as the SNPare “hallmark” tumor suppressor genes. “Hallmark” tumor suppressor genesare a small list of very-well validated tumor suppressor genes that aremore likely to be mutated early in tumor development. These genes were:TP53, PTEN, APC, MLL3, MLL2, VHL, CDKN2A, and RB1. A 1 in this columnindicates that one of these hallmark TSGs exists on the same chromosomeas the SNP in question and is significantly mutated in the tumor typeanalyzed.

The term “gistic_deletion_n_peaks: The number of GISTIC peaks on thechromosome on which the SNP falls. A higher number suggests (loosely)that there are more selective forces driving loss of genetic material onthis chromosome.

The term “gistic_deletion_best_q_value: The lowest GISTIC q-value forgenomic loss on the chromosome on which the SNP falls. A very lowq-value suggests that there is a significant selective pressure to losegenomic material somewhere on the chromosome.

The term “proportion_of_patients_eligible: The estimated proportion ofpatients who would have i) germline heterozygosity of the SNP and ii)LOH of the SNP in tumor. The estimate of the proportion of patients withgermline heterozygosity of the SNP assumes Hardy-Weinberg equilibrium,using the equation proportion heterozygote=2pq. Where p is the globalallelic fraction of the SNP and q=1−p.

The term “proportion_of_patients_eligible_max_ethnicity_targeted: Theestimated proportion of patients who would have i) germlineheterozygosity of the SNP and ii) LOH of the SNP in tumor. The estimateof the proportion of patients with germline heterozygosity of the SNPassumes Hardy-Weinberg equilibrium, using the equation proportionheterozygote=2pq. Where p is the maximum population-restricted allelicfraction of the SNP and q=1−p. For example, in some cases the populationused might be African and in some cases it might be South Asian.

The term “cumulative_score: A score that quantifies the degree to whicha SNP is a good candidate for an iCAR target. Scores range from 0 totheoretical 1. For more information on the calculation of this score,please see the section titled “Cumulative score to rank candidate SNPs.”

Example 1. Assessment of Rate of LOH of HLA Genes Across CancersIntroduction

A therapeutic strategy is proposed to address vulnerabilities incurredby genomic loss in cancer cells. The proposed strategy uses acombination of activating-CAR T-cells (aCAR) and inhibitory-CAR T-cells(iCAR) to more safely target tumors that have lost genomic segmentsencoding cell-membrane proteins heterozygous for the maternal andpaternal alleles (i.e., with polymorphic protein coding changes).

iCARs can decrease off-tumor toxicity of CAR-T therapy withoutdecreasing anti-tumor efficacy if the target of the iCAR is expressedonly by non-tumor tissues. One such scenario in which iCAR targets areexpressed only by non-tumor cells occurs when the iCAR antigen isencoded by a portion of the genome that has been deleted in tumor cells.One gene family that is highly polymorphic and known to be expressed onall cells is HLA.

The HLA proteins are nearly universally expressed by mammalian cells toallow for the presentation of non-self antigens to cells of the immunesystem. HLA genes also tend to be quantitatively highly expressed,making them more amenable to therapeutic targeting. The RNA expressionof the HLA genes is higher than 99.3 percent of other protein codinggenes in the genome (FIG. 4). The mean tissue expression of HLA genesand their genomic locations is included in Table 3 as well as thelengthy table provided herewith on CD-ROM.

The goal of this section is to identify cancer types in which the HLAgene undergoes frequent deletion. Secondary analyses include attempts toidentify drivers of genomic loss at the HLA locus.

We executed a detailed plan for identifying cancers with selectivepressures that drove frequent copy-loss of HLA genes (FIG. 5).

Frequency of HLA Loss Across Tumor Types Using ABSOLUTE Data:

We used copy number profiles from the TCGA that had been processed bythe ABSOLUTE algorithm to assess ground-truth estimates of the rate ofallelic loss of HLA-A. Publicly available ABSOLUTE segmented copy-numberdata were downloaded from(https://www.synapse.org/#!Synapse:syn1710464.2)¹. The ABSOLUTEalgorithm outputs the integer copy level of each allelic segment withina single cancer genome. In the case of loss of a single copy ofchromosome 6 (harboring the HLA locus), then the allelic copy numberswould be: 1 for the retained segment and 0 for the segment that waslost. In the case of copy-neutral loss of heterozygosity, then theretained segment would have copy number 2 and the lost segment wouldhave copy number 0. Publically available copy number data processed byABSOLUTE were available for 12 tumor types (Table 4). Lung squamous cellcarcinoma (LUSC) had the highest frequency of HLA-A LOH compared to theother tumor types (FIG. 6). Uterine/endometrial cancers (UCEC) had thelowest frequency of HLA-A LOH of all the evaluable tumors (AML sampleswere not included due to ABSOLUTE data not being available). Of 588deletions of the HLA-A gene, none had an intragenic breakpoint (FIG. 7).Most deletions of HLA-A genes encompassed large portions of thechromosome (FIG. 8). While ABSOLUTE copy number data were not availablefor AML samples, manual inspection of the relative copy number data inthese samples revealed no deletions (FIG. 11).

Validation of Relative Copy Number Data Compared to ABSOLUTE Copy NumberData:

We sought to obtain the frequency of LOH of as many tumor types as werepublicly available. However, these data had not been processed byABSOLUTE and the raw data to process by ABSOLUTE are not publiclyavailable. Instead, we used relative copy number data on 32 tumor typesfrom TCGA (FIG. 13). These data were downloaded from cbioportal(cbioportal.org/data_sets.jsp). The relative copy number data wereobtained from Affymetrix SNP 6.0 arrays of tumor samples.

In order to determine whether accurate estimates of LOH could beobtained from relative copy number data, we computed the rate of LOHwith relative data for the tumors that had already had LOH data fromABSOLUTE. These data consisted of a segmented copy number file. Eachsegment is assigned a relative copy-ratio. The copy ratio is defined asthe log of the ratio of density of signal in tumor compared to thematched normal (in Affymetrix arrays). The normalization to a matchedcontrol (usually from peripheral blood) helps to remove any germlinecopy-number variants from mistakenly being interpreted as somatic. Asegment is said to have undergone genomic loss if the relative copynumber of that genomic segment is below a given threshold. For example,if the relative copy number of segment 321 is −0.4 and the threshold forcopy-loss is −0.3, then segment 321 is said to have undergone copy-lossand because we lack direct allelic information, it is said to haveundergone LOH as well.

We first attempted to determine the optimal copy number cutoff forlabeling relative copy number segments as having undergone LOH. Theconcordance of ABSOLUTE and relative copy number estimates of LOH washighest with a cutoff of −0.1 for relative copy number (Table 5 and FIG.9). This threshold also happens to be the threshold used by the TCGAcopy number group to define copy-loss in the TCGA Tumorscape portal(http://portals.broadinstitute.org/tcga/home). The correlation betweenthe fraction of individuals with HLA-A LOH in relative data vs ABOSLUTEdata was 0.55. This reasonably high correlation enabled us to moveforward with the analysis of all tumor types with relative copy numberdata available.

Fraction of Patients with HLA-LOH Across 32 Tumor Types Using RelativeCopy Number Data

The portion of patients that had LOH of HLA-A was computed for all 32tumors available from TCGA (FIG. 10A; COAD and READ were analyzedtogether). The tumor with the highest rate of HLA-A LOH was kidneychromophobe cancer. The tumor with the lowest rate of HLA-A LOH wasuveal melanoma (Table 6). To ensure that the rate of LOH we had derivedin these analyses was robust to small perturbation of genomic position,we analyzed the rate of LOH of the upstream and downstream genes ofHLA-A to see if their rate of HLA-LOH was similar to HLA-A. As expected,the rate of LOH of the upstream and downstream genes, HLA-G and ZNRD1respectively was exactly the same as for HLA-A. (FIG. 3 A-C). These datademonstrate that the HLA-A LOH calls are robust to small deviations ingenomic position. Next, we sought to determine whether the other HLAgenes (A, B, C) had similar rates of LOH compared to HLA-A. These genesall fall within a 1.3 Mb region on chromosome 6p. In genomic distance,this is a small region. We repeated the HLA-A analysis on HLA-B andHLA-C. The pattern of LOH was nearly identical between all three HLAgenes across the 32 tumors analyzed (FIG. 10A-C).

Addition of Selection Pressure to HLA-A LOH Rates

Intratumoral genomic heterogeneity is a recently appreciated feature ofnearly all human cancers analyzed to date^(2,3). Therapies targeted togenetic alterations only present in a fraction of tumor cells may onlyaffect the tumor cells harboring said alterations. An iCAR strategy thattargets antigens not present on tumor cells may protect some tumor cellsfrom aCAR attack if the antigen is not clonally deleted. We thereforesought to identify tumors in which HLA genes were likely to undergoclonal LOH. LOH that occurs early in evolution is likely to be driven byselective forces in tumor initiation and/or maintenance. We thereforelooked for tumor suppressors on chromosome 6 (harboring HLA locus) inthree ways. First, we looked for genes that were significantly mutatedon chromosome 6 in each of the tumor types assessed⁴. The spreadsheetreports the genes with significant mutation on chromosome 6 under the“chr6_mutsig_sig_genes” column.

Second, we looked for regions of significantly deleted genes, signifyinglikely deleted tumor suppressors. We used the results of GISTIC2.0 runon these data. The spreadsheet reports the number of GISTIC deletionpeaks on chromosome 6 (q<0.25) and the lowest q-value of these deletionpeaks. Generally, the more GISTIC deletion peaks and the lower theq-value, the stronger the selection pressure. However, it is alsopossible to have the scenario where one very strong GISTIC peakpredominates and the number of peaks would be small, but thesignificance of the driver is for certain. In general, the lowestq-value should be the strongest correlate of tumor suppressor driverpresence on a given chromosome.

Third, we overlapped the set of genes that were significantly mutated ineach tumor with a list of known tumor suppressor genes to determine ifany of the mutated genes was likely to drive loss of chromosome 6⁵. Wewere able to identify two tumor types with possible mutational drivers.In adrenocortical carcinoma, the DAXX gene was significantly mutated(q=0.0571) and in Diffuse Large B Cell Lymphoma, the TNFAIP3 gene wassignificantly mutated (q=0.00278). DAXX encodes a histone chaperone,mutations of which are associated with longer telomeres inadrenocortical carcinoma⁶. TNFAIP3 encodes a negative regulator ofNF-kappaB signaling. Mutations of this gene occurring in DLBCL have beenshown to therefore increase NF-kappaB signaling⁷.

TABLE 3 Genomic loci analyzed for LOH. Genomic coordinates are in thehg19 human genome assembly. RNA Start End Expression Gene ProteinChromosome Position Position (RPKM) HLA-A HLA-A 6 29941260 29945884226.6 HLA-B HLA-B 6 31353872 31357188 422.4 HLA-C HLA-C 6 3126874931272130 193.4

TABLE 4 Tumor types with ABSOLUTE data Number of Number Samples TCGA ofFinished Disease Name Abbreviation Samples ABSOLUTE Bladder urothelialBLCA 138 90 carcinoma Breast invasive BRCA 880 750 carcinoma Colonadenocarcinoma COAD 422 349 Glioblastoma GBM 580 485 multiforme Head andNeck HNSC 310 270 squamous cell carcinoma Kidney renal clear cell KIRC497 373 carcinoma Acute Myeloid LAML 200 0 Leukemia Lung adenocarcinomaLUAD 357 292 Lung squamous cell LUSC 344 261 carcinoma Ovarian serous OV567 457 cystadenocarcinoma Rectum READ 164 147 adenocarcinoma Uterinecorpus UCEC 498 378 endometrial carcinoma

TABLE 5 Correlation (Pearson) of LOH rate by relative copy number datavs ABSOLUTE copy number data. Correlation peaks for a threshold value of−0.1. Deletion threshold Correlation (r²) 0 0.01 −0.05 0.49 −0.1 0.55−0.15 0.53 −0.2 0.46 −0.25 0.44 −0.3 0.21 −0.35 0.10 −0.4 0.07 −0.450.09 −0.5 0.08

TABLE 6 Numbers and rates of LOH for all 32 cancers in the TCGA dataset.TCGA Total samples Number with Fraction Abbreviation (n) LOH (n) withLOH KICH 66 57 0.863636364 ACC 90 46 0.511111111 PAAD 184 51 0.277173913KIRP 288 73 0.253472222 LUSC 501 124 0.24750499 SARC 257 63 0.245136187ESCA 184 45 0.244565217 KIRC 528 98 0.185606061 BLCA 408 73 0.178921569OV 579 96 0.165803109 THYM 123 20 0.162601626 HNSC 522 81 0.155172414CESC 295 45 0.152542373 STAD 441 66 0.149659864 BRCA 1080 1590.147222222 DLBC 48 7 0.145833333 LUAD 516 65 0.125968992 COADREAD 61677 0.125 GBM 577 72 0.124783362 TGCT 150 18 0.12 CHOL 36 4 0.111111111MESO 87 9 0.103448276 UCS 56 5 0.089285714 UCEC 539 31 0.057513915 LGG513 24 0.046783626 PRAD 492 19 0.038617886 SKCM 104 4 0.038461538 LIHC370 14 0.037837838 PCPG 162 3 0.018518519 THCA 499 9 0.018036072 UVM 800 0 LAML 0 0 NA

Based on the above, we concluded that HLA region LOH is a common eventin many tumors, however and the percentage of LOH varies between tumortypes. Therefore, HLA genes are good candidates for iCAR targets.

REFERENCES FOR EXAMPLE 1

-   1. Zack T I, Schumacher S E, Carter S L, Cherniack A D, Saksena G,    Tabak B, Lawrence M S, Zhsng C Z, Wala J, Mermel C H, Sougnez C,    Gabriel S B, Hernandez B, Shen H, Laird P W, Getz G, Meyerson M,    Beroukhim R. Pan-cancer patterns of somatic copy number alteration.    Nature genetics. 2013; 45:1134-1140-   2. Gibson W J, Hoivik E A, Halle M K, Taylor-Weiner A, Cherniack A    D, Berg A, Holst F, Zack T I, Werner H M, Staby K M, Rosenberg M,    Stefansson I M, Kusonmano K, Chevalier A, Mauland K K, Trovik J,    Krakstad C, Giannakis M, Hodis E, Woie K, Bjorge L, Vintermyr O K,    Wala J A, Lawrence M S, Getz G, Carter S L, Beroukhim R, Salvesen    H B. The genomic landscape and evolution of endometrial carcinoma    progression and abdominopelvic metastasis. Nature genetics. 2016;    48:848-855-   3. Gerlinger M, Rowan A J, Horswell S, Math M, Larkin J, Endesfelder    D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, Varela    I, Phillimore B, Begum S, McDonald N Q Butler A, Jones D, Raine K,    Latimer C, Santos C R, Nohadani M, Eklund A C, Spencer-Dene B, Clark    G, Pickering L, Stamp G, Gore M, Szallasi Z, Downward J, Futreal P    A, Swanton C. Intratumor heterogeneity and branched evolution    revealed by multiregion sequencing. The New England journal of    medicine. 2012; 366:883-892-   4. Lawrence M S, Stojanov P, Mermel C H, Robinson J T, Garraway L A,    Golub T R, Meyerson M, Gabriel S B, Lander E S, Getz G. Discovery    and saturation analysis of cancer genes across 21 tumour types.    Nature. 2014; 505:495-501-   5. Vogelstein B, Papadopoulos N, Velculescu V E, Zhou S, Diaz L A,    Jr., Kinzler K W. Cancer genome landscapes. Science. 2013;    339:1546-1558-   6. Zheng S, Cherniack A D, Dewal N, Moffitt R A, Danilova L, Murray    B A, Lerario A M, Else T, Knijnenburg T A, Ciriello G, Kim S, Assie    G, Morozova O, Akbani R, Shih J, Hoadley K A, Choueiri T K, Waldmann    J, Mete O, Robertson A G, Wu H T, Raphael B J, Shao L, Meyerson M,    Demeure M J, Beuschlein F, Gill A J, Sidhu S B, Almeida M Q Fragoso    M, Cope L M, Kebebew E, Habra M A, Whitsett T G, Bussey K J, Rainey    W E, Asa S L, Bertherat J, Fassnacht M, Wheeler D A, Cancer Genome    Atlas Research N, Hammer G D, Giordano T J, Verhaak R G W.    Comprehensive pan-genomic characterization of adrenocortical    carcinoma. Cancer cell. 2016; 29:723-736-   7. Compagno M, Lim W K, Grunn A, Nandula S V, Brahmachary M, Shen Q,    Bertoni F, Ponzoni M, Scandurra M, Califano A, Bhagat G, Chadburn A,    Dalla-Favera R, Pasqualucci L. Mutations of multiple genes cause    deregulation of nf-kappab in diffuse large b-cell lymphoma. Nature.    2009; 459:717-721

Example 2. Genome-Wide Identification of Germline Alleles that EncodeExpressed Cell-Surface Proteins that Undergo Loss of HeterozygosityIntroduction

Inhibitory-CAR-T cells can decrease off-tumor toxicity of CAR-T therapywithout decreasing anti-tumor efficacy if the target of the iCAR isexpressed only by non-tumor tissues. One such scenario in which iCARtargets will be expressed only by non-tumor cells is where the iCARantigen is encoded by a portion of the genome that has been deleted intumor cells. The goal of this section of the workflow is to identifysuch alleles.

Allele Identification:

We used the Exome Aggregation Consortium (ExAC) database as an input tothe analysis (exac.broadinstitute.org). The ExAC database is acompilation of exomes from various population-level sequencing studiestotaling 60,706 exomes¹. ExAC contains information about each variantincluding the number of counts of the reference allele compared to thealternative allele (allelic frequency). The allelic frequencyinformation is extended to subpopulations within the database asdetailed in Table 7.

TABLE 7 Subpopulations within the ExAC database. Population PopulationNumber of ancestry Abbreviation Individuals African AFR 5,203 Latino AMR5,789 East Asian EAS 4,327 Finnish FIN 3,307 Non-Finnish European NFE33,370 South Asian SAS 8,256 Other OTH 454 Note: Not all positions ingenome have sufficient coverage in exome such that all individuals inthis table are represented. Source: http://exac.broadinstitute.org/faq.

The following filters were applied to variants from the ExAC database:i) the variant must affect the amino acid composition of the encodedprotein ii) the variant must have a minor allele frequency of greaterthan 0.05 (5%) in at least one of the populations in Table 6. Theanalysis corrected for scenarios where the minor allele had an allelefraction greater than 0.5 (50%). If more than three alleles at a sitewere observed, then the most prevalent substitution was used (thesesites are often sites of sequencing error and should be interpreted withcaution).

A SNP was counted as having an impact on the composition of the proteinif any of the SNP produced any of the following variant classes:‘missense_variant’, ‘inframe_deletion’, ‘start_lost’, ‘stop_gained’,‘inframe_insertion’, ‘stop_retained_variant’, ‘frameshift_variant’,‘stop_lost’, ‘coding_sequence_variant’, ‘protein_altering_variant’. Theanalysis started with 9,362,319 variants and 29,904 variants passedthese two filters. These variants fell in 10,302 genes. All allelesmatching these two filters were included in the analysis.

Identification of Expressed Genes:

We used the Genotype-Tissue Expression (GTEX) database v6p (dbGaPAccession phs000424.v6.p1) for the identification of genes that areexpressed in various tissue types (https://gtexportal.org/home/)². TheGTEX database consists of RNA-sequencing of 8,555 human samples fromdiverse healthy tissue types. Several annotations were obtained fromthis database. First, we determined the average expression of each geneacross all tissues. The mean expression for each gene was calculated bytaking the per-tissue median expression data and computing the mean ofthese values across tissues. These data were obtained from the fileGTEx_Analysis_v6p_RNA-seq_RNA-SeQCv1.1.8_gene_median_rpkm.gct fromavailable at https://gtexportal.org/home/datasets.

The mean expression of each gene corresponding to each tumor type wasalso included. To obtain these data we created a mapping of tumor typesto corresponding normal tissues. For example, the pancreatic cancer TCGAdata would be annotated with pancreas tissue from GTEX. In some cases,the mapping was more approximate. For example, the glioblastomasexpression data were mapped from all tissues annotated as brain in GTEX.A table with these mappings (titled tcga_disease_tissue_lookup.txt) isattached Several measures were computed to assess the homogeneity oroverexpression of each gene in each tissue/tumor type. For each tumortype, a cohen's-D score was computed to establish possibleover-expression of the gene. Genes overexpressed in particular tissues,are likely to be good aCAR targets. Conversely, we measured the standarddeviation of gene expression across tissues and compared this to themean expression across all tissues. When this ratio was low, the gene isevenly expressed across all tissues. Genes with even expression acrossall tissues are likely to be better iCAR targets.

A gene was called “universally expressed” if it met the followingcriteria: (i) the mean express across tissues was greater than 10 RPKM.(ii) The tissues with the least expression had an RPKM greater than 1.(iii) The ratio of the standard deviation in median RPKM across tissuescompared to the mean RPKM was less than 1. Only 1,092 genes wereannotated as universally expressed.

Candidates were selected only based on the UniProt annotation. Fortransmembrane proteins, there is usually clear prediction for segmentsof the protein that are extracellular.

Table 8 presents a list of 1167 good candidate genes identified by theabove method having extracellular polymorphic epitopes sorted accordingto chromosome location.

Annotation of Alleles Impact of Allele on Protein Function:

For an iCAR to effectively recognize only cancer cells that have lostone allele of a membrane protein, the protein's structure out to besufficiently different based on which allele is encoded. Severalmeasures were taken to quantify the effect of each SNP on the resultingprotein. First, the reported SNP variant class (e.g. missense, nonsense)was reported in the column ‘consequence’. The effect on the consensusprotein translation was included in the ‘protein_consequence’ (e.g.,p.Arg482G1n) column. The SIFT algorithm attempts to predict whether aprotein variant will have an effect on the protein structure, andtherefore function⁶. The score can range from 0 (deleterious) to 1(benign). SIFT scores (version sift5.2.2) were included for every SNPfor which a score was available. Scores are not available for frameshiftmutations for example. PolyPhen (v2.2.2) was also used to makeprediction on the possibility that a variant may affect proteinstructure and function. The Polyphen algorithm reports scores in theopposite manner of SIFT, with a score of 0 corresponding to benign and ascore of 1 corresponding to deleterious.

One classic measure of an amino acids substitution probability ofinducing a structural change is to use the BLOSUM62 substitution matrix.We downloaded the BLOSUM62 matrix fromhttps://www.ncbi.nlm.nih.gov/IEB/ToolBox/C_DOC/lxr/source/data/BLOSUM62.Each SNP was annotated with the BLOSUM62 score corresponding to itssubstitution.

Classification of Allele as Falling in the Extracellular Portion of theProtein:

For an iCAR to recognize an allele, the allele must fall on theextracellular portion of the protein. For each SNP, we extracted theposition of the amino acid affected in the consensus translation andcompared this to domains annotated as extracellular from the Uniprotdatabase. The Uniprot database was downloaded fromwww.uniprot.org/downloads. Many false negatives are possible due to alack of characterization of the domains of all proteins. A total of 3288SNPs in 1167 genes were annotated as extracellular (Table 8).

Annotations of Peptide Context of SNP:

The peptide context of the alleles analyzed will likely matter whentrying to generate antibodies that recognize these sequences. We includefor reference the 10 amino acids preceding and flanking the amino acidencoded by the SNP (21 amino acid sequence total). The uniprot databasewas used for the consensus amino acid sequence. We annotate anyconflicts where the uniprot database sequence did not match the aminoacid encoded by either SNP at the predicted position, so as not toinclude any false sequences. These 21 amino acid sequences could beuseful as input to B-cell epitope prediction programs such as Bepipred.

Cancer-Specific Annotations: Proportion of Tumors Undergoing LOH

Finding patients whose tumors could benefit from the proposed therapywould require an iCAR target would be a SNP that undergoes loss ofheterozygosity (LOH) in a large fraction of tumors. Segments copy numberfiles were downloaded from the cbio cancer genomics portalhttp://www.cbioportal.org/⁸. As an example, the proportion of uvealmelanoma tumors undergoing LOH for all SNPs is shown in FIG. 12.

Potential Driver Alterations on Chromosomes Harboring Candidate SNPs

One possible mechanism of resistance genomically targeted therapy is ifone of the intended genomic alterations in only present in a fraction ofthe cancer cells. One mechanism to attempt to identify targets likely tobe present in the earliest stages of tumor development is to identifydriver events for each tumor. The most frequent mechanism of tumorsuppressor gene inactivation is mutation and subsequent LOH of thenon-mutated chromosome. We attempted to find driver genes, particularlytumor suppressor genes (TSGs) likely to undergo this process in eachtumor type. We used the results of MUTSIG 2.0 run on all tumors in thisanalysis to identify genes significantly mutated in each tumor type. Weannotated whether or not one of the genes that was significantly mutatedwas included in a list of “hallmark” tumor suppressor genes includingTP53, PTEN, APC, MLL3, MLL2, VHL, CDKN2A, RB1. Finally, the list ofdriver genes, TSG, and “hallmark” TSGs were annotated onto a SNP if theyfell on the same chromosome as the SNP.

While mutations in driver genes that subsequently undergo LOH is onemechanism that may mark events likely to occur early in tumor evolution,focal deletion of genomic segments containing a tumor suppressor gene isanother. We used the GISTIC algorithm to identify regions of DNA thatundergo genomic deletion at a rate higher than average. The GISTICalgorithm identifies “peaks” of statistical significance alongchromosome arms that suggest a negative selective pressure on theseregions. For each SNP, we recorded the number of deletion peaks on thechromosome that the SNP fell on. We also recorded the lowest q-value ofany of these peaks. A lower q-value suggests stronger selectivepressure.

Cumulative Score to Rank Candidate SNPs:

In an effort to provide a continuous “score” for the candidate SNPs, wecombined several different metrics that should be associated with betterSNP candidates. The score consists of the product of the percentile rankof each of the following:

1. proportion of tumors with LOH at that SNP (higher is better) 2.prevalence of the allele (higher is better) 3. ratio of the standarddeviation of expression values across tissues to the median (lower isbetter, more consistent) 4. whether or not there is a tumor suppressorgene on the chromosome (having one is better than not having one)

To illustrate, we will calculate the score for a theoretical SNP. Ifonly 32% of the SNPs had a tumor suppressor gene on the chromosome, thenthe percentile rank for having one would be 0.68. If the allele had aminor allele fraction of 0.49 (where 0.5 is the highest possible), thenthe percentile rank would be 0.99. If the rate of LOH was 0.10, and 75%of SNPs had more LOH than that, then the percentile rank would be 0.25.If the ratio of standard deviation of expression values across tissuesto the median for the gene harboring this SNP was 1.3 and that is betterthan 90% of other genes, then the percentile rank is 0.9. The totalscore for this SNP would then be 0.68*0.99*0.25*0.9=0.15.

Any SNP with a score greater than 0.4 was considered “top-hit”.

TABLE 8 Exemplary iCAR Targets Chr. No. Gene 1 ABCA4 1 ADAM30 1 ASTN1 1C1orf101 1 CACNA1S 1 CATSPER4 1 CD101 1 CD164L2 1 CD1A 1 CD1C 1 CD244 1CD34 1 CELSR2 1 CHRNB2 1 CLCA2 1 CLSTN1 1 CR1 1 CR2 1 CRB1 1 CSF3R 1CSMD2 1 ECE1 1 ELTD1 1 EMC1 1 EPHA10 1 EPHA2 1 ERMAP 1 FCAMR 1 FCER1A 1FCGR1B 1 FCGR2A 1 FCGR2B 1 FCGR3A 1 FCRL1 1 FCRL3 1 FCRL4 1 FCRL5 1FCRL6 1 GJB4 1 GPA33 1 GPR157 1 GPR37L1 1 GPR88 1 HCRTR1 1 IGSF3 1 IGSF91 IL22RA1 1 ITGA10 1 KIAA1324 1 KIAA2013 1 LDLRAD2 1 LEPR 1 LRIG2 1 LRP81 LRRC52 1 LRRC8B 1 LRRN2 1 LY9 1 MR1 1 MUC1 1 MXRA8 1 NCSTN 1 NFASC 1NOTCH2 1 NPR1 1 NTRK1 1 OPN3 1 OR10J1 1 OR10J4 1 OR10K1 1 OR10R2 1OR10T2 1 OR10X1 1 OR11L1 1 OR14A16 1 OR14I1 1 OR14K1 1 OR2AK2 1 OR2C3 1OR2G2 1 OR2G3 1 OR2L2 1 OR2M7 1 OR2T1 1 OR2T12 1 OR2T27 1 OR2T29 1 OR2T31 OR2T33 1 OR2T34 1 OR2T35 1 OR2T4 1 OR2T5 1 OR2T6 1 OR2T7 1 OR2T8 1OR2W3 1 OR6F1 1 OR6K2 1 OR6K3 1 OR6K6 1 OR6N1 1 OR6P1 1 OR6Y1 1 PEAR1 1PIGR 1 PLXNA2 1 PTCH2 1 PTCHD2 1 PTGFRN 1 PTPRC 1 PTPRF 1 PVRL4 1 RXFP41 S1PR1 1 SCNN1D 1 SDC3 1 SELE 1 SELL 1 SELP 1 SEMA4A 1 SEMA6C 1 SLAMF71 SLAMF9 1 SLC2A7 1 SLC5A9 1 TACSTD2 1 TAS1R2 1 TIE1 1 TLR5 1 TMEM81 1TNFRSF14 1 TNFRSF1B 1 TRABD2B 1 USH2A 1 VCAM1 1 ZP4 2 ABCG5 2 ALK 2ASPRV1 2 ATRAID 2 CD207 2 CHRNG 2 CLEC4F 2 CNTNAP5 2 CRIM1 2 CXCR1 2DNER 2 DPP10 2 EDAR 2 EPCAM 2 GPR113 2 GPR148 2 GPR35 2 GPR39 2 IL1RL1 2ITGA4 2 ITGA6 2 ITGAV 2 LCT 2 LHCGR 2 LRP1B 2 LRP2 2 LY75 2 MARCO 2MERTK 2 NRP2 2 OR6B2 2 PLA2R1 2 PLB1 2 PROKR1 2 PROM2 2 SCN7A 2 SDC1 2TGOLN2 2 THSD7B 2 TMEFF2 2 TMEM178A 2 TPO 2 TRABD2A 3 ACKR2 3 ALCAM 3ANO10 3 ATP13A4 3 CACNA1D 3 CACNA2D2 3 CACNA2D3 3 CASR 3 CCRL2 3 CD200 3CD200R1 3 CD86 3 CD96 3 CDCP1 3 CDHR4 3 CELSR3 3 CHL1 3 CLDN11 3 CLDN183 CLSTN2 3 CSPG5 3 CX3CR1 3 CXCR6 3 DCBLD2 3 DRD3 3 EPHB3 3 GABRR3 3 GP53 GPR128 3 GPR15 3 GPR27 3 GRM2 3 GRM7 3 HEG1 3 HTR3C 3 HTR3D 3 HTR3E 3IGSF11 3 IL17RC 3 IL17RD 3 IL17RE 3 IL5RA 3 IMPG2 3 ITGA9 3 ITGB5 3KCNMB3 3 LRIG1 3 LRRC15 3 LRRN1 3 MST1R 3 NAALADL2 3 NRROS 3 OR5AC1 3OR5H1 3 OR5H14 3 OR5H15 3 OR5H6 3 OR5K2 3 OR5K3 3 OR5K4 3 PLXNB1 3PLXND1 3 PRRT3 3 PTPRG 3 ROBO2 3 RYK 3 SEMA5B 3 SIDT1 3 SLC22A14 3SLC33A1 3 SLC4A7 3 SLITRK3 3 STAB1 3 SUSD5 3 TFRC 3 TLR9 3 TMEM44 3TMPRSS7 3 TNFSF10 3 UPK1B 3 VIPR1 3 ZPLD1 4 ANTXR2 4 BTC 4 CNGA1 4 CORIN4 EGF 4 EMCN 4 ENPEP 4 EPHA5 4 ERVMER34-1 4 EVC2 4 FAT1 4 FAT4 4 FGFRL14 FRAS1 4 GPR125 4 GRID2 4 GYPA 4 GYPB 4 KDR 4 KIAA0922 4 KLB 4 MFSD8 4PARM1 4 PDGFRA 4 RNF150 4 TENM3 4 TLR1 4 TLR10 4 TLR6 4 TMEM156 4TMPRSS11A 4 TMPRSS11B 4 TMPRSS11E 4 TMPRSS11F 4 UNC5C 5 ADAM19 5 ADRB2 5BTNL3 5 BTNL8 5 BTNL9 5 C5orf15 5 CATSPER3 5 CD180 5 CDH12 5 CDHR2 5COL23A1 5 CSF1R 5 F2RL2 5 FAM174A 5 FAT2 5 FGFR4 5 FLT4 5 GABRA6 5GABRG2 5 GPR151 5 GPR98 5 GRM6 5 HAVCR1 5 HAVCR2 5 IL31RA 5 IL6ST 5 IL7R5 ITGA1 5 ITGA2 5 KCNMB1 5 LIFR 5 LNPEP 5 MEGF10 5 NIPAL4 5 OR2V1 5OR2Y1 5 OSMR 5 PCDH1 5 PCDH12 5 PCDHA1 5 PCDHA2 5 PCDHA4 5 PCDHA8 5PCDHA9 5 PCDHB10 5 PCDHB11 5 PCDHB13 5 PCDHB14 5 PCDHB15 5 PCDHB16 5PCDHB2 5 PCDHB3 5 PCDHB4 5 PCDHB5 5 PCDHB6 5 PCDHGA1 5 PCDHGA4 5 PDGFRB5 PRLR 5 SEMA5A 5 SEMA6A 5 SGCD 5 SLC1A3 5 SLC22A4 5 SLC22A5 5 SLC36A3 5SLC6A18 5 SLC6A19 5 SLCO6A1 5 SV2C 5 TENM2 5 TIMD4 5 UGT3A1 6 BAI3 6BTN1A1 6 BTN2A1 6 BTN2A2 6 BTN3A2 6 BTNL2 6 CD83 6 DCBLD1 6 DLL1 6 DPCR16 ENPP1 6 ENPP3 6 ENPP4 6 EPHA7 6 GABBR1 6 GABRR1 6 GCNT6 6 GFRAL 6 GJB76 GLP1R 6 GPR110 6 GPR111 6 GPR116 6 GPR126 6 GPR63 6 GPRC6A 6 HFE 6HLA-A 6 HLA-B 6 HLA-C 6 HLA-DPA1 6 HLA-DPB1 6 HLA-DQA1 6 HLA-DQA2 6HLA-DQB1 6 HLA-DQB2 6 HLA-DRB1 6 HLA-DRB5 6 HLA-E 6 HLA-F 6 HLA-G 6IL20RA 6 ITPR3 6 KIAA0319 6 LMBRD1 6 LRFN2 6 LRP11 6 MAS1L 6 MEP1A 6MICA 6 MICB 6 MUC21 6 MUC22 6 NCR2 6 NOTCH4 6 OPRM1 6 OR10C1 6 OR12D2 6OR12D3 6 OR14J1 6 OR2B2 6 OR2B6 6 OR2J1 6 OR2W1 6 OR5V1 6 PKHD1 6 PTCRA6 RAET1E 6 RAET1G 6 ROS1 6 SDIM1 6 SLC22A1 6 SLC44A4 6 TAAR2 6 TREM1 6TREML1 6 TREML2 7 AQP1 7 CD36 7 CDHR3 7 CNTNAP2 7 DPP6 7 EGFR 7 EPHA1 7EPHB6 7 ERVW-1 7 GHRHR 7 GJC3 7 GPNMB 7 GRM8 7 HYAL4 7 KIAA1324L 7 LRRN37 MET 7 MUC12 7 MUC17 7 NPC1L1 7 NPSR1 7 OR2A12 7 OR2A14 7 OR2A2 7OR2A25 7 OR2A42 7 OR2A7 7 OR2AE1 7 OR2F2 7 OR6V1 7 PILRA 7 PKD1L1 7PLXNA4 7 PODXL 7 PTPRN2 7 PTPRZ1 7 RAMP3 7 SLC29A4 7 SMO 7 TAS2R16 7TAS2R4 7 TAS2R40 7 TFR2 7 THSD7A 7 TMEM213 7 TTYH3 7 ZAN 7 ZP3 8 ADAM188 ADAM28 8 ADAM32 8 ADAM7 8 ADAM9 8 CDH17 8 CHRNA2 8 CSMD1 8 CSMD3 8DCSTAMP 8 FZD6 8 GPR124 8 NRG1 8 OR4F21 8 PKHD1L1 8 PRSS55 8 SCARA3 8SCARA5 8 SDC2 8 SLC10A5 8 SLC39A14 8 SLC39A4 8 SLCO5A1 8 TNFRSF10A 8TNFRSF10B 9 ABCA1 9 AQP7 9 C9orf135 9 CA9 9 CD72 9 CNTNAP3 9 CNTNAP3B 9ENTPD8 9 GPR144 9 GRIN3A 9 IZUMO3 9 KIAA1161 9 MAMDC4 9 MEGF9 9 MUSK 9NOTCH1 9 OR13C2 9 OR13C3 9 OR13C5 9 OR13C8 9 OR13C9 9 OR13D1 9 OR13F1 9OR1B1 9 OR1J2 9 OR1K1 9 OR1L1 9 OR1L3 9 OR1L6 9 OR1L8 9 OR1N1 9 OR1N2 9OR1Q1 9 OR2S2 9 PCSK5 9 PLGRKT 9 PTPRD 9 ROR2 9 SEMA4D 9 SLC31A1 9 TEK 9TLR4 9 TMEM2 9 VLDLR 10 ABCC2 10 ADAM8 10 ADRB1 10 ANTXRL 10 ATRNL1 10C10orf54 10 CDH23 10 CDHR1 10 CNNM2 10 COL13A1 10 COL17A1 10 ENTPD1 10FGFR2 10 FZD8 10 GPR158 10 GRID1 10 IL15RA 10 IL2RA 10 ITGA8 10 ITGB1 10MRC1 10 NPFFR1 10 NRP1 10 OPN4 10 PCDH15 10 PKD2L1 10 PLXDC2 10 PRLHR 10RGR 10 SLC29A3 10 SLC39A12 10 TACR2 10 TCTN3 10 TSPAN15 10 UNC5B 10VSTM4 11 AMICA1 11 ANO3 11 APLP2 11 C11orf24 11 CCKBR 11 CD248 11 CD4411 CD5 11 CD6 11 CDON 11 CLMP 11 CRTAM 11 DCHS1 11 DSCAML1 11 FAT3 11FOLH1 11 GDPD4 11 GDPD5 11 GRIK4 11 HEPHL1 11 HTR3B 11 IFITM10 11 IL10RA11 KIRREL3 11 LGR4 11 LRP4 11 LRP5 11 LRRC32 11 MCAM 11 MFRP 11 MPEG1 11MRGPRE 11 MRGPRF 11 MRGPRG 11 MRGPRX2 11 MRGPRX3 11 MRGPRX4 11 MS4A4A 11MTNR1B 11 MUC15 11 NAALAD2 11 NAALADL1 11 NCAM1 11 NRXN2 11 OR10A2 11OR10A5 11 OR10A6 11 OR10D3 11 OR10G4 11 OR10G7 11 OR10G8 11 OR10G9 11OR10Q1 11 OR10S1 11 OR1S1 11 OR2AG1 11 OR2AG2 11 OR2D2 11 OR4A15 11OR4A47 11 OR4A5 11 OR4A8P 11 OR4C11 11 OR4C13 11 OR4C15 11 OR4C16 11OR4C3 11 OR4C46 11 OR4C5 11 OR4D6 11 OR4D9 11 OR4S2 11 OR4X1 11 OR51E111 OR51L1 11 OR52A1 11 OR52E1 11 OR52E2 11 OR52E4 11 OR52E6 11 OR52I1 11OR52I2 11 OR52J3 11 OR52L1 11 OR52N1 11 OR52N2 11 OR52N4 11 OR52W1 11OR56B1 11 OR56B4 11 OR5A1 11 OR5A2 11 OR5AK2 11 OR5AR1 11 OR5B17 11OR5B3 11 OR5D14 11 OR5D16 11 OR5D18 11 OR5F1 11 OR5I1 11 OR5L2 11 OR5M1111 OR5M3 11 OR5P2 11 OR5R1 11 OR5T2 11 OR5T3 11 OR5W2 11 OR6A2 11 OR6T111 OR6X1 11 OR8A1 11 OR8B12 11 OR8B2 11 OR8B3 11 OR8B4 11 OR8D1 11 OR8D211 OR8H1 11 OR8H2 11 OR8H3 11 OR8I2 11 OR8J1 11 OR8J2 11 OR8J3 11 OR8K111 OR8K3 11 OR8K5 11 OR8U1 11 OR9G1 11 OR9G4 11 OR9Q2 11 P2RX3 11 PTPRJ11 ROBO3 11 SIGIRR 11 SLC22A10 11 SLC3A2 11 SLC5A12 11 SLCO2B1 11 SORL111 ST14 11 SYT8 11 TENM4 11 TMEM123 11 TMPRSS4 11 TMPRSS5 11 TRPM5 11TSPAN18 11 ZP1 12 ANO4 12 AVPR1A 12 CACNA2D4 12 CD163 12 CD163L1 12 CD2712 CD4 12 CLEC12A 12 CLEC2A 12 CLEC4C 12 CLEC7A 12 CLECL1 12 CLSTN3 12GPR133 12 GPRC5D 12 ITGA7 12 ITGB7 12 KLRB1 12 KLRC2 12 KLRC3 12 KLRC412 KLRF1 12 KLRF2 12 LRP1 12 LRP6 12 MANSC1 12 MANSC4 12 OLR1 12 OR10AD112 OR10P1 12 OR2AP1 12 OR6C1 12 OR6C2 12 OR6C3 12 OR6C4 12 OR6C6 12OR6C74 12 OR6C76 12 OR8S1 12 OR9K2 12 ORAI1 12 P2RX4 12 P2RX7 12 PTPRB12 PTPRQ 12 SCNN1A 12 SELPLG 12 SLC38A4 12 SLC5A8 12 SLC6A15 12 SLC8B112 SLCO1B1 12 SLCO1B7 12 SSPN 12 STAB2 12 TAS2R10 12 TAS2R13 12 TAS2R2012 TAS2R30 12 TAS2R31 12 TAS2R42 12 TAS2R43 12 TAS2R46 12 TAS2R7 12TMEM119 12 TMEM132B 12 TMEM132C 12 TMEM132D 12 TMPRSS12 12 TNFRSF1A 12TSPAN8 12 VSIG10 13 ATP4B 13 ATP7B 13 FLT3 13 FREM2 13 KL 13 PCDH8 13SGCG 13 SHISA2 13 SLC15A1 13 SLITRK6 13 TNFRSF19 14 ADAM21 14 BDKRB2 14C14orf37 14 CLEC14A 14 DLK1 14 FLRT2 14 GPR135 14 GPR137C 14 JAG2 14LTB4R2 14 MMP14 14 OR11G2 14 OR11H12 14 OR11H6 14 OR4K1 14 OR4K15 14OR4K5 14 OR4L1 14 OR4N2 14 OR4N5 14 OR4Q2 14 SLC24A4 14 SYNDIG1L 15ANPEP 15 CD276 15 CHRNA7 15 CHRNB4 15 CSPG4 15 DUOX1 15 DUOX2 15 FAM174B15 GLDN 15 IGDCC4 15 ITGA11 15 LCTL 15 LTK 15 LYSMD4 15 MEGF11 15 NRG415 OCA2 15 OR4F4 15 OR4M2 15 OR4N4 15 PRTG 15 RHCG 15 SCAMP5 15 SEMA4B15 SEMA6D 15 SLC24A1 15 SLC28A1 15 TRPM1 15 TYRO3 16 ATP2C2 16 CACNA1H16 CD19 16 CDH11 16 CDH16 16 CDH3 16 CDH5 16 CNGB1 16 CNTNAP4 16 GDPD316 GPR56 16 GPR97 16 IL4R 16 ITFG3 16 ITGAL 16 ITGAM 16 ITGAX 16 KCNG416 MMP15 16 MSLNL 16 NOMO1 16 NOMO3 16 OR2C1 16 PKD1 16 PKD1L2 16 SCNN1B16 SEZ6L2 16 SLC22A31 16 SLC5A11 16 SLC7A6 16 SPN 16 TMC5 16 TMC7 16TMEM204 16 TMEM219 16 TMEM8A 17 ABCC3 17 ACE 17 AOC3 17 ASGR2 17C17orf80 17 CD300A 17 CD300C 17 CD300E 17 CD300LG 17 CHRNB1 17 CLEC10A17 CNTNAP1 17 CPD 17 CXCL16 17 FAM171A2 17 GCGR 17 GLP2R 17 GP1BA 17GPR142 17 GUCY2D 17 ITGA2B 17 ITGA3 17 ITGAE 17 ITGB3 17 KCNJ12 17LRRC37A 17 LRRC37A2 17 LRRC37A3 17 LRRC37B 17 MRC2 17 NGFR 17 OR1A2 17OR1D2 17 OR1G1 17 OR3A1 17 OR3A2 17 OR4D1 17 OR4D2 17 RNF43 17 SCN4A 17SDK2 17 SECTM1 17 SEZ6 17 SLC26A11 17 SPACA3 17 TMEM102 17 TMEM132E 17TNFSF12 17 TRPV3 17 TTYH2 17 TUSC5 18 APCDD1 18 CDH19 18 CDH20 18 CDH718 COLEC12 18 DCC 18 DSC1 18 DSG1 18 DSG3 18 DYNAP 18 MEP1B 18 PTPRM 18SIGLEC15 18 TNFRSF11A 19 ABCA7 19 ACPT 19 BCAM 19 C19orf38 19 C19orf5919 C5AR1 19 CATSPERD 19 CATSPERG 19 CD320 19 CD33 19 CD97 19 CEACAM1 19CEACAM19 19 CEACAM21 19 CEACAM3 19 CEACAM4 19 CLEC4M 19 DLL3 19 EMR1 19EMR2 19 EMR3 19 ERVV-1 19 ERVV-2 19 FAM187B 19 FCAR 19 FFAR3 19 FPR1 19GFY 19 GP6 19 GPR42 19 GRIN3B 19 ICAM3 19 IGFLR1 19 IL12RB1 19 IL27RA 19KIR2DL1 19 KIR2DL3 19 KIR2DL4 19 KIR3DL1 19 KIR3DL2 19 KIR3DL3 19KIRREL2 19 KISS1R 19 LAIR1 19 LDLR 19 LILRA1 19 LILRA2 19 LILRA4 19LILRA6 19 LILRB1 19 LILRB2 19 LILRB3 19 LILRB4 19 LILRB5 19 LINGO3 19LPHN1 19 LRP3 19 MADCAM1 19 MAG 19 MEGF8 19 MUC16 19 NCR1 19 NOTCH3 19NPHS1 19 OR10H1 19 OR10H2 19 OR10H3 19 OR10H4 19 OR1I1 19 OR2Z1 19OR7A10 19 OR7C1 19 OR7D4 19 OR7E24 19 OR7G1 19 OR7G2 19 OR7G3 19 PLVAP19 PTGIR 19 PTPRH 19 PTPRS 19 PVR 19 SCN1B 19 SHISA7 19 SIGLEC10 19SIGLEC11 19 SIGLEC12 19 SIGLEC5 19 SIGLEC6 19 SIGLEC8 19 SIGLEC9 19SLC44A2 19 SLC5A5 19 SLC7A9 19 TARM1 19 TGFBR3L 19 TMC4 19 TMEM91 19TMPRSS9 19 TNFSF14 19 TNFSF9 19 TRPM4 19 VN1R2 19 VSIG10L 19 VSTM2B 20ABHD12 20 ADAM33 20 ADRA1D 20 APMAP 20 ATRN 20 CD40 20 CD93 20 CDH22 20CDH26 20 CDH4 20 FLRT3 20 GCNT7 20 GGT7 20 JAG1 20 LRRN4 20 NPBWR2 20OCSTAMP 20 PTPRA 20 PTPRT 20 SEL1L2 20 SIGLEC1 20 SIRPA 20 SIRPB1 20SIRPG 20 SLC24A3 20 SLC2A10 20 SSTR4 20 THBD 21 CLDN8 21 DSCAM 21 ICOSLG21 IFNAR1 21 IFNGR2 21 IGSF5 21 ITGB2 21 KCNJ15 21 NCAM2 21 TMPRSS15 21TMPRSS2 21 TMPRSS3 21 TRPM2 21 UMODL1 22 CACNA1I 22 CELSR1 22 COMT 22CSF2RB 22 GGT1 22 GGT5 22 IL2RB 22 KREMEN1 22 MCHR1 22 OR11H1 22 P2RX622 PKDREJ 22 PLXNB2 22 SCARF2 22 SEZ6L 22 SSTR3 22 SUSD2 22 TMPRSS6 22TNFRSF13C X ATP6AP2 X ATP7A X EDA2R X FMR1NB X GLRA4 X GPR112 X GUCY2F XHEPH X P2RY10 X P2RY4 X PLXNA3 X PLXNB3 X VSIG4 X XG

REFERENCES FOR EXAMPLE 2

-   1. Lek M, Karczewski K J, Minikel E V, Samocha K E, Banks E, Fennell    T, O'Donnell-Luria A H, Ware J S, Hill A J, Cummings B B, Tukiainen    T, Birnbaum D P, Kosmicki J A, Duncan L E, Estrada K, Zhao F, Zou J,    Pierce-Hoffman E, Berghout J, Cooper D N, Deflaux N, DePristo M, Do    R, Flannick J, Fromer M, Gauthier L, Goldstein J, Gupta N, Howrigan    D, Kiezun A, Kurki M I, Moonshine A L, Natarajan P, Orozco L, Peloso    G M, Poplin R, Rivas M A, Ruano-Rubio V, Rose S A, Ruderfer D M,    Shakir K, Stenson P D, Stevens C, Thomas B P, Tiao G, Tusie-Luna M    T, Weisburd B, Won H H, Yu D, Altshuler D M, Ardissino D, Boehnke M,    Danesh J, Donnelly S, Elosua R, Florez J C, Gabriel S B, Getz G,    Glatt S J, Hultman C M, Kathiresan S, Laakso M, McCarroll S,    McCarthy M I, McGovern D, McPherson R, Neale B M, Palotie A, Purcell    S M, Saleheen D, Scharf J M, Sklar P, Sullivan P F, Tuomilehto J,    Tsuang M T, Watkins H C, Wilson J G, Daly M J, MacArthur D G, Exome    Aggregation C. Analysis of protein-coding genetic variation in    60,706 humans. Nature. 2016; 536:285-291-   2. Consortium G T. Human genomics. The genotype-tissue expression    (gtex) pilot analysis: Multitissue gene regulation in humans.    Science. 2015; 348:648-660-   3.-   6. Ng P C, Henikoff S. Sift: Predicting amino acid changes that    affect protein function. Nucleic acids research. 2003; 31:3812-3814-   7-   8. Cerami E, Gao J, Dogrusoz U, Gross B E, Sumer S O, Aksoy B A,    Jacobsen A, Byrne C J, Heuer M L, Larsson E, Antipin Y, Reva B,    Goldberg A P, Sander C, Schultz N. The cbio cancer genomics portal:    An open platform for exploring multidimensional cancer genomics    data. Cancer discovery. 2012; 2:401-404

Example 3. DNA Sequencing Analysis for Verification of HLA LOH in KichSamples Library Preparation and Sequencing

Purpose—based on the in-silico analysis, KICH cancer was chosen as thefirst tumor type for wet verification of the HLA LOH prediction. The aimwas to identify the HLA genotype for each pateinet based on DNA derivedfrom normal tissue, and then to analyse the HLA allotype in the cancertisse in an attempt to identify loss of one of the HLA alleles.

For that matter, HLA allotype was determined for DNA derived from 6frozen matche KICH samples (Normal and Cancer) RC-001-RC003, TNEABAll,TNEABNWE, 2rDFRAUB, 2RDFRNQG, IOWT5AVJ, IOWT5N74. In addition, two DNAmatched samples OG-001-OG-002 (Normal and Cancer) were also analysed. ADNA library was prepared sequence analysis was conducted in order toidentify the sample's HLA typing. DNA was extracted from 6 frozenmatched KICH samples (Normal and tumor) and a library was prepared asdescribed below.

TruSight HLA Sequencing libraries were prepared using TruSight® HLA v2Sequencing Panel (Illumina, San Diego, Calif., U.S.A.) at GenotypicTechnology Pvt. Ltd., Bangalore, India.

Briefly, HLA Amplicons were generated using the primers provided in theTruSight HLA Sequencing Kit. Amplicons were confirmed on Agarose Gelfollowed by cleanup of the amplicons using Aample Purification Beadsprovided in the kit. Amplicons were normalized and fragmented byTagmentation reaction. Post Tagmentation different amplicons of eachindividual sample were pooled and proceeded for enrichment PCR.Barcoding of the samples was done during enrichment PCR using Nextera XTIndex Kit v2 (Illumina). Final PCR product was purified using SamplePurification Beads followed by quality control check of the libraries.Libraries were quantified by Qubit fluorometer (Thermo FisherScientific, MA, USA) and its fragment size distribution was analyzed onAgilent Bioanalyzer.

Illumina Adapter Sequences:

5′ - AATGATACGGCGACCACCGAGATCTACAC [i5] TCGTCGGCAG CGTC5′ - CAAGCAGAAGACGGCATACGAGAT [i7] GTCTCGTGGGCTCGG[i5, i7] - Unique dual index sequence to identifysample-specific sequencing data

The table below depict the HLA genotype of the above samples.

As seen below, we can infer the lost allele from the analysis, forexample, patient #RC001 exhibits loss of HLA-A30 allele in the tumorsamples and becomes hemizygout to HLA-32; patient #RC003 lost HLA-1 inthe tumor sample and becomes hemizygout to HLA-30. The identified lostallele will determine the relevant iCAR for each patient. Cases wheretumor samples were contaminated with normal cells, could exhibit clearHLA allele loss in this method.

TABLE 9 HLA genotype of the matched KICH samples Sample_ID HLA-A HLA-BHLA-C OG_001_NA1_NORMAL 02:06:01:— 7:02:01 03:04:01:— 24:02:01:—15:01:01:— 07:02:01:— OG_001_TUM_TUMOR 02:06:01:— 7:02:01 03:04:01:—24:02:01:— 15:01:01:— 07:02:01: OG_002_NAT_NORMAL 02:01:01:— 15:01:01:—3:03:01 24:02:01:— 55:01:01 X OG_002_TUM_TUMOR 02:01:01:— 15:01:01:—3:03:01 24:02:01:— 55:01:01 X RC_002_NAT_A_NORMAL 03:01:01:— 7:02:0106:02:01:— 68:02:01:— 58:02:01 7:18:00 RC_002_TUM_A_TUMOR 03:01:01:—7:02:01 06:02:01:— 68:02:01:— 58:02:01 7:18:00 RC_003_NAT_A_NORMAL01:01:01:01 7:02:01 07:01:01:— 30:04:01 49:01:01 07:02:01:—RC_003_TUM_A_TUMOR 30:04:01 7:02:01 07:01:01:— X 49:01:01 07:02:01:—RC_001_NAT_B_NORMAL 30:04:01 53:01:01 04:01:01:— 32:01:01 58:02:0106:02:01:— RC_001_TUM_B_TUMOR 32:01:01 53:01:01 04:01:01:— X 58:02:0106:02:01:— 2RDFRAUB_Tumor 03:01:01:— 7:02:01 07:02:01:— 32:01:0138:01:01 12:03:01:— SO_7534_SET3_2RDFRNQG_Normal 03:01:01:— 7:02:0107:02:01:— 32:01:01 38:01:01 12:03:01:— IOWT5AVJ_Tumor 34:02:0115:03:01:— 02:10:01:— 68:01:01:— 81:01:00 8:04:01 IOWT5N 74_Normal34:02:01 15:03:01:— 02:10:01:— 68:01:01:— 81:01:00 8:04:01 TNEAB1L_Tumor02:01:01:— 8:01:01 03:04:01:— 03:01:01:— 40:01:02 07:01:01:—TNEABNWE_Normal 02:01:01:— 8:01:01 03:04:01:— 03:01:01:— 40:01:0207:01:01:— X—no variant reads

Exome Sequencing

In addition to the HLA sequencing, we also performed exome sequencing inorder to confirm HLA-LOH and to identify additional LOH events acrossthe genome

The Illumina paired end raw reads (150×2, HiSeq) were quality checkedusing FastQC. Illumina raw reads were processed by Trim Galore softwarefor adapter clipping and low quality base trimming using parameters ofminimum read length 50 bp and minimum base quality 30. The processedreads were aligned to the reference human genome (hg19) using Bowtie2.Then aligned .bam files for each of the samples were processed to getthe final PCR duplicate removed .bam files and alignment quality waschecked using Qualimap.

Variants were identified using SAMtools and BCFtools. In this case,joint genotyping is done to identify variants in each pair of samples(each normal and tumor pair). Therefore, for each pair a merged .vcf isgenerated. Potential variants are identified from each of these merged.vcf files using read depth threshold >20 and mapping quality >30. Fromeach pair of the filtered merged .vcf, sample-wise .vcf files weregenerated. The filtered variants were further annotated for genes,protein change and the impact of the variations using Variant Studio.

The below table describes the extent of chromosome loss for the abovesamples. RC001, RC002 and RC003 exhibit extensive chromosome lossincluding chromosome 6 which codes for HLA genes, hence, for thesesamples, HLA can be used as iCAR target, in addition to many othertargets coded on chromosomes 1, 2, 3, 4 (for RC002), 5, 6, 8 (forRC003), 9 (RC001, RC002), 10 (RC001, RC003), 11 (RC003), 13 (RC001,RC003), 14 (RC002), 17 (RC001, RC003), 19 (RC001), 21 (RC001, RC003),22(RC001, RC002).

TABLE 10 chromosome loss Chr RC001 RC002 RC003 OG001 OG002 2RD IOW TNE 1++ ++ ++ 2 ++ + ++ + 3 ++ ++ + 4 ++ 5 ++ ++ 6 ++ + ++ + 7 8 ++ 9 ++ ++++ 10 ++ ++ 11 ++ 12 + 13 ++ ++ 14 + + 15 + 16 17 ++ ++ 18 19 ++ 20 21++ ++ 22 ++ ++ ++ + LOH (Chr loss) for about 50% of the cells ++ LOH(Chr loss) for almost 100% of the cells

For RC001, FIG. 14 depict the loss of a chromosomal region adjacent tothe tumor suppressor protein TP53, coded on chromosome 17. Genes codedon chromosome 17 which were identified as iCAR targets can be used totreat patient RC001.

Abbreviations: ADP, adenosine diphosphate; ALL, acute lymphoblasticleukemia; AML, acute myelogenous leukemia; APRIL, aproliferation-inducing ligand; BAFF, B cell activation factor of the TNFfamily; BCMA, B cell maturation antigen; BCR, B cell receptor; BM, bonemarrow; CAIX, carbonic anhydrase IX; CAR, chimeric antigen receptor;CEA, carcinoembryonic antigen; CLL, chronic lymphocytic leukemia; CNS,central nervous system; CSPG4, chondroitin sulfate proteoglycan 4; DC,dendritic cell; ECM, extracellular matrix; EGFR, epidermal growth factorreceptor; EGFRvIII, variant III of the EGFR; EphA2,erythropoietin-producing hepatocellular carcinoma A2; FAP, fibroblastactivation protein; FR-α, folate receptor-alpha; GBM, glioblastomamultiforme; GPI, glycophosphatidylinositol; H&N, head and neck; HL,Hodgkin's lymphoma; Ig, immunoglobulin; L1-CAM, L1 cell adhesionmolecule; MM, multiple myeloma; NB, neuroblastoma; NF-KB, nuclearfactor-KB; NHL, non-Hodgkin's lymphoma; NK, natural killer; NKG2D-L,NKG2D ligand; PBMC, peripheral blood mononuclear cell; PC, plasma cell;PLL, prolymphocytic leukemia; PSCA, prostate stem cell antigen; PSMA,prostate-specific membrane antigen; RCC, renal cell carcinomas; ROR1,receptor tyrosine kinase-like orphan receptor 1; TCL, T cellleukemia/lymphoma; Th2, T helper 2; TNBC, triple-negative breast cancer;TNFR, tumor necrosis factor receptor; VEGFR-2, vascular endothelialgrowth factor-2.

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Example 4. Verification of LOH at the Protein Level

LOH can be detected at the protein level by differential staining ofnormal vs. tumor cell samples using allele specific antibodies. Forexample, verification of HLA-LOH in cancer samples, can be done usingcommercial HLA antibodies specific to the patient's HLA allotype. Table11 below details an example for available allele-specific antibodies,which can be used.

Samples will be subjected to immuno-histochemistry (IHC) staining asdescribed in the IHC protocol below.

TABLE 11 Allele-specific anti-HLA antibodies Antibody ManufacturerAnti-human HLA-A2 APC (BB7.2) eBiosciences Anti-human HLA-A2 PE-cy7(BB7.2) eBiosciences Anti-human HLA-A3 FITC (GAP A3) eBiosciencesAnti-human HLA-A3 PE (GAP A3) eBiosciences Mouse Anti-HLA Class 1Antigen A25, A32 US Biological Antibody HLA Class 1 Antigen A30, A31MyBioSource mouse anti-human HLA-B7-PE (BB7.1) Millipore HLA-A2 antibody(BB7.2) Novus HLA B7 antibody (BB7.1) Novus Mouse anti-humanHLA-B27-FITC (clone Millipore HLA.ABC.m3)

IHC Protocol Frozen Tissues Samples—

Frozen tissues are often fixed in a formalin-based solution, andembedded in OCT (Optimal Cutting Temperature compound), that enablescryosectioning of the sample. Tissues in OCT are kept frozen at −80° C.Frozen blocks are removed from −80° C. prior to sectioning, equilibratedin cryostat chamber, and cut to thin sections (often 5-15 μm thick).Sections are mounted on a histological slide. Slides can be stored at−20° C. to −80° C. Prior to

IHC staining, slides are thawed at room temperature (RT) for 10-20 min.

Paraffin-Embedded Tissues—

Tissues are embedded in a Formaldehyde Fixative Solution. Prior toaddition of the paraffin wax, tissues are dehydrated by gradualimmersion in increasing concentrations of ethanol (70%, 90%, 100%) andxylene for specific times and durations at RT. Then, the tissues areembedded in paraffin wax.

The paraffin-embedded tissues are cut in a microtome to a 5-15 μm thicksections, floated in a 56° C. water bath, and mounted on a histologicalslide. Slides can be kept at RT.

Prior to IHC staining, paraffin-embedded sections require a rehydrationstep. REHYDRATION—sections are rehydrated by immersion in xylene (2×10min), followed by decreasing concentrations of ethanol—100%×2, each for10 min 95% ethanol—5 min 70% ethanol—5 min 50% ethanol—5 min Rinsing indH2O.

Immunofluorescence Detection:

Protocol:

-   -   1. Rehydrate slides in wash buffer (PBSX1) for 10 min. Drain the        wash buffer.    -   2. Perform antigen retrieval—if needed (heat-induced antigen        retrieval or enzymatic retrieval).    -   3. For intracellular antigens, perform permeabilization—incubate        the slides in 0.1% triton X-100 in PBSX1 for 10 min at RT.    -   4. BLOCKING—Block the tissue in blocking buffer for 30 min. at        RT. Blocking buffer depends on the detection method, usually 5%        animal serum in PBSX1, or 1% BSA in PBSX1    -   5. PRIMARY ANTIBODY—Dilute primary antibody in incubation buffer        (e.g., 1% BSA, 1% donkey serum in PBS, other incubation buffers        can also be used), according to antibody manufacturer        instructions. Incubate the tissue in diluted primary antibody at        4° C. overnight. The primary antibody may be a monoclonal        anti-HLA-A, anti-HLA-B or anti-HLA-C allele-specific antibody as        detailed above.        -   If a conjugated primary antibody is used, protect from            light, and proceed to step 8.        -   As a negative control, incubate the tissue with incubation            buffer only, with no primary antibody.        -   Also, perform isotype matched control of the monoclonal            antibody used in the experiment.    -   6. 6. WASH—wash slides in wash buffer—3×5-15 min.    -   7. 7. SECONDARY ANTIBODY—Dilute secondary antibody in incubation        buffer according to antibody manufacturer instructions. Incubate        the tissue in diluted secondary antibody for 30-60 min at RT.        Protect from light.    -   8. 8. WASH—wash slides in wash buffer—3×5-15 min.    -   9. 9. DAPI staining—Dilute DAPI incubation buffer (˜300 nM—3        μM). Add 300 μl of DAPI solution to each section. Incubate at RT        for 5-10 min.    -   10. 10. WASH—wash slide once with ×1 PBS.    -   11. 11. Mount with an antifade mounting media.    -   12. 12. Keep slides protected from light.    -   13. 13. Visualize slides using a fluorescence microscope.

Chromogenic Detection:

Protocol:

-   -   1. 1. Rehydrate slides in wash buffer (PBSX1) for 10 min. Drain        the wash buffer.    -   2. 2. Perform antigen retrieval—if needed—see above.    -   3. 3. For HRP reagents, block endogenous peroxidase activity        with 3.0% hydrogen peroxide in methanol for at least 15 min.    -   4. 4. Wash the sections by immersing them in dH2O for 5 min.    -   5. 5. For intracellular antigens, perform        permeabilization—incubate the slides in 0.1% triton X-100 in        PBSX1 for 10 min at RT.    -   6. 6. BLOCKING—Block the tissue in blocking buffer for 30 min.        at RT. Blocking buffer depends on the detection method, usually        5% animal serum in PBSX1, or 1% BSA in PBSX1.    -   7. 7. PRIMARY ANTIBODY—Dilute primary antibody in incubation        buffer (e.g., 1% BSA, 1% donkey serum in PBS, other incubation        buffers can also be used), according to antibody manufacturer        instructions. Incubate the tissue in diluted primary antibody at        4° C. overnight    -   8. 8. WASH—wash slides in wash buffer—3×5-15 min.    -   9. 9. SECONDARY ANTIBODY—Incubate the tissue in HRP-conjugated        secondary antibody for 30-60 min at RT.    -   10. 10. WASH—wash slides in wash buffer 3×5-15 min.    -   11. 11. Add ABC-HRP reagent according to manufacturer        guidelines. Incubate at RT for 60 min.    -   12. 12. Prepare DAB solution (or other chromogen) according to        manufacturer guidelines, and apply to tissue sections. The        chromogenic reaction turns the epitope sites brown (usually few        seconds—10 minutes). Proceed to the next step when the intensity        of the signal is appropriate for imaging    -   13. 13. WASH—wash slides in wash buffer—3×5-15 min.    -   14. 14. Wash slides in dH2O—2×5-15 min.    -   15. 15. Nuclei staining—add Hematoxylin solution. Incubate at RT        for 5 min.    -   16. 16. Dehydrate tissue sections—95% ethanol—2×2 min. 100%        ethanol—2×2 min. Xylene—2×2 min.    -   17. 17. Mount with an antifade mounting media    -   18. 18. Visualize slides using a bright-field illumination

Example 5. CAR-T Design and Construction

The purpose of the study is to create a synthetic receptor which willinhibit the on-target ‘off-tumor’ effect of CAR-T therapy. To thatextent a library of CAR constructs composed of activating and inhibitoryCARs was established.

The first set of constructs included an inhibitory CAR directed at HLAtype I sequence (HLA-A2) and an activating CAR directed at tumor antigen(CD19). The next set of constructs to be used for the sake of proof ofconcept, includes activating CAR sequences directed at CD19 and aninhibitory CAR sequences directed at CD20. Additional constructsdirected at target antigens identified by future bioinformatics analysiswill be constructed. Target candidates will be prioritized according toset forth criteria (exemplary criteria include but are not limited to,target expression pattern, target expression level, antigenicity andmore). A CD19 aCAR, CD20 iCAR, and HLA-A2 iCAR have been constructed, asdescribed in FIGS. 15 and 21.

iCAR constructs were designed and synthesized using commercial DNAsynthesis. The transmembrane and intracellular domains up to the firstannotated extracellular domain of PD-1 (amino acid 145-288) was fuseddownstream to HLA-A2 scFv (DNA sequence coding for HLA-A2, was retrievedfrom hybridoma BB7.2, (ATCC cat #: HB-82), producing anti HLA-A2).

Similar constructs with CTLA4 (amino acids 161-223) or with othersequences derived from additional negative immune regulators (forexample 2B4, LAG-3 and BTLA-4) will be designed and their signalingsequences will be fused downstream to the HLA-A2 scFv.

For iCAR detection and sorting, a reporter gene (e.g., eGFP) wasintegrated downstream to the iCAR sequence via IRES sequences andfollowed by an antibiotic resistance gene (i.e., hygromycin) separatedby P2A sequence, as illustrated in FIG. 15.

For the aCAR construct, CD19 scFV was fused to 2nd generation CARconstruct composed of CD8 hinge sequence followed by CD28 transmembraneand 41BB co-stimulation 1 and CD3. Additional aCAR constructs composedof other signaling or structural element will also be designed andconstructed (e.g. CD28 hinge, CD28 signaling domain or both CD28 and41BB signaling domains). For aCAR detection and sorting, RFP a reportergene was integrated downstream to the aCAR sequence via IRES sequencesfollowed by antibiotic resistance gene (Puromycin resistance) separatedby P2A sequence (FIG. 15).

Both aCAR and iCAR sequences were cloned into lentivirus transfer vectorand then used for viral particle production using HEK-293T packagingcells.

Example 6. Production of Effector Cells

To study the effect of the iCAR constructs on modulating CD19 CARactivation, recombinant Jurkat effector cells were constructed asdetailed in Table 12 below. Jurkat (ATCC TIB152), a CD4+ T-cell line andJurkat-NFAT (a Jurkat cell-line purchased from BPS Biosciences,engineered to express a firefly luciferase protein, under the control ofNFAT response elements) were transduced using retronectin-coated(Takara) lentiviral vector bound plates or in the presence of polybrene.Transduced cells were further subjected to antibiotic selection to yieldthe cell-lines described in Table 12. Following selection, the cellswere subjected to flow cytometry analysis to verify the expression ofthe reporter protein coded on each construct.

TABLE 12 recombinant effector cell-lines Recombinant Construct 1Construct 2 effector cell-line Parental Cell (aCAR-RFP) (iCAR-GFP) CD19aCAR Jurkat Jurkat CD19 aCAR — CD19aCAR/HLA- Jurkat CD19 aCAR HLA-A2iCAR A2 iCAR Jurkat HLA-A2 iCAR Jurkat — HLA-A2 iCAR JurkatCD19aCAR/CD20 Jurkat CD19 aCAR CD20 iCAR iCAR Jurkat CD20 iCAR JurkatJurkat — CD20 iCAR CD19 aCAR Jurkat- Jurkat-NFAT CD19 aCAR — NFATCD19aCAR/HLA- Jurkat-NFAT CD19 aCAR HLA-A2 iCAR A2 iCAR Jurkat- NFATHLA-A2 iCAR Jurkat-NFAT — HLA-A2 iCAR Jurkat-NFAT CD19aCAR/CD20Jurkat-NFAT CD19 aCAR CD20 iCAR iCAR Jurkat-NFAT CD20 iCAR Jurkat-Jurkat-NFAT — CD20 iCAR NFAT

In addition, activated T-cells, derived from peripheral blood obtainedfrom healthy donors will be transduced with viral particles coding foraCAR, iCAR or both, at different multiplicity of infection (MOI). FACSselection based on reporter gene expression will be used for sorting andselection of cell population expressing different level of aCAR, iCAR orboth.

Example 7. Preparation of Target Cells

An in-vitro recombinant system was established for testing thefunctionality of iCAR constructs in inhibiting the activity of the aCARtowards off-target cells. For this purpose, target cells expressing theaCAR epitope, iCAR epitope or both were produced. The recombinant cellsexpressing the aCAR epitope represent the ‘on-target’ ‘on-tumor’ cells,while the cells expressing both aCAR and iCAR epitopes represent the‘on-target’ ‘off-tumor’ healthy cells.

As our first iCAR/aCAR set is based on HLA-A2 and CD19 respectively,recombinant cells expressing HLA-A2 or CD19 or both were produced, bytransfecting cell line (e.g., Hela, ATCC CRM-CCL-2,Hela-Luciferase—GenTarget SC032-Bsd or Raji-ATCC CCL-86) with expressionvectors coding for these genes.

For detection of recombinant HLA A-2 expression, Myc tag was inserted.For the second iCAR/aCAR set comprised of CD20 iCAR/CD19 aCAR,recombinant cells expressing CD20 or CD19 or both were constructed(target cells are detailed in Table 15).

TABLE 13 Target cell-lines Parental Target Target Set# cell protein 1protein 2 Purpose Modeling 1 Raji CD19 None A model for cancer cellsOn-tumor expressing endogenous CD19 Raji CD19 HLA-A2 A model for normalcells Off-tumor expressing endogenous CD19; recombinant HLA-A2 Thp 1None HLA_A2 A model for normal cells Negative control expressingendogenous HLA-A2 and negative to CD19 2 Hela HLA-A2 None A model fornormal cells Negative control expressing endogenous HLA-A2 and negativeto CD19 Hela HLA-A2 CD19 A model for normal cells Off-tumor expressingrecombinant CD19; HLA-A2 4 Hela CD19 None A model for cancer cellsOn-tumor expressing recombinant CD19 Hela CD19 CD20 A model for normalcells Off-tumor expressing recombinant CD19; CD20 Hela CD20 None A modelfor normal cells Negative control expressing endogenous CD20 andnegative to CD19 3 Hela- HLA-A2 None Negative control to be Negativecontrol Luciferase used in killing assay Hela- HLA-A2 CD19 A model fornormal cells Off-tumor Luciferase expressing recombinant CD19; HLA-A2(killing assay) 5 Hela- CD19 None A model for cancer cells On-tumorLuciferase expressing recombinant CD19 (killing assay) Hela- CD19 CD20 Amodel for normal cells Off-tumor Luciferase expressing recombinant CD19;CD20 (killing assay) Hela- CD20 None Negative control (killing Negativecontrol Luciferase assay)

Assays—

iCAR's inhibitory effect will be tested both in-vitro and in-vivo.

In the in-vitro assays, we will focus on measuring cytokine secretionand cytotoxicity effects, while in-vivo, we will evaluate the iCARinhibition and protection to ‘on-target off-tumor’ xenografts. We willlimit T-cells lacking iCAR from contaminating the results by sortingT-cells to be iCAR/aCAR double positive using reporter genes. As anegative control for iCAR blocking activity, we may use T-cellstransduced with CAR lacking the scFv domain (i.e. mock transduction).

Example 8. In Vitro Assays

Luciferase Cytotoxic T lymphocyte (CTL) Assay

Assay will be performed using Hela-Luc recombinant target cellsdescribed above, engineered to express firefly luciferase and one or twoCAR target antigens. In-vitro luciferase assay will be performedaccording to the Bright-Glo Luciferase assay manufacture's protocol(Promega) and bioluminescence as a readout.

T-cells (transduced with both iCAR and pCAR or iCAR and aCAR or aCAR ormock CAR) will be incubated for 24-48 hrs. with the recombinant targetcells expressing HLA-A2 or CD19 or both HLA-A2 and CD19 or CD20 or bothCD20 and CD19 in different effector to target ratios. Cell killing willbe quantified with the Bright-Glo Luciferase system.

The ‘off-tumor’ cytotoxicity is optimized by sorting transduced T-cellspopulation according to iCAR/aCAR expression level or by selecting subpopulation of recombinant target cells according to their CD19, HLA-A2or CD20 expression level. To test whether iCAR transduced T-cells candiscriminate between the ‘on-tumor’ and ‘off-tumor’ cells in vitro, wewill test the killing effect of transduced T-cells incubated with a mixof ‘on-tumor’ and ‘off-tumor’ cells at a ratio of 1:1 and more. The‘on-tumor’ recombinant cells will be distinguished from the ‘off-tumor’recombinant cells by Luciferase expression (only one cell populationwill be engineered to express the luciferase gene at a given time).Killing will be quantified after 24-48 hrs of co-incubation.

Caspase 3 Activity Assay—Detection of CTL Induced Apoptosis by anAnti-Activated Caspase 3 (CASP3).

One of the pathways by which cytotoxic T-cells kill target cells is byinducing apoptosis through the Fas ligand. Sequential activation ofcaspases plays a significant role in the execution-phase of cellapoptosis. Cleavage of pro-caspase 3 to caspase 3 results inconformational change and expression of catalytic activity. The cleavedactivated form of caspase 3 can be specifically recognized by amonoclonal antibody.

Transduced T-cells will be co-cultured for 2-4 hrs with either‘on-tumor’ or ‘off-tumor’ recombinant cells, previously labeled withCFSE or other cell tracer dye (e.g. CellTrace Violet). Following cellpermeabilization and fixation by an inside staining kit (e.g. Miltenyior BD bioscience) activated CASP3 will be detected by specific antibodystaining (BD bioscience), and apoptotic target cells will be detectedand quantified by flow cytometry.

Time Lapse Microscopy CTL

Transduced T-cells will be incubated with either ‘on-tumor’ or‘off-tumor’ cells for up to 5 days. Time lapse microscopy will be usedto visualize killing. Alternatively, flow cytometry analysis usingviable cell number staining and CountBright beads (Invitrogen) fordetermining target cells number at end-point time will be conducted.

In order to demonstrate the effectiveness of aCAR/iCAR transducedT-cells in discerning targets in vitro, each recombinant target cells(‘on-tumor’ or ‘off-tumor’) is labeled with a different reporter protein(e.g. GFP and mCherry). Transduced T-cells (Effector cells) will beco-incubated with a mix of recombinant cells expressing one or twotarget antigens (Target cells) at different E/T ratios. Each cell-line'sfate will be followed by microscopy imaging.

Cytokine Release

Upon T-cell activation, the cells secrete cytokines which can bequantified and used for evaluating T-cell activation and inhibition.Cytokines can be detected intracellularly by flow cytometry or bymeasurement of the secreted proteins in the medium by ELISA orCytometric Bead Array (CBA).

Quantitation of Secreted Cytokines by ELISA

Following co-cultivation of transduced T-cells (Jurkat, or primaryT-cells) expressing iCAR or aCAR or both aCA and iCAR with modifiedtarget cells, expressing iCAR or aCAR or both aCAR and iCAR antigens ontheir cell surface, conditioned medium will be collected, and cytokine'sconcentration will be measured by cytokine ELISA (IL-2, INFγ and orTNFα) according to the manufacture instruction (e.g. BioLegened orsimilar), and by Cytometric Bead Array (Miltenyi or similar).

iCAR Specific Inhibition as Measured by IL-2 ELISA

Jurkat CD19 aCAR and Jurkat CD19 aCAR/HLA-A2 iCAR effector cells wereco-cultured with Raji, Raji-HLA-A2 and Thp1 target cells and thecorresponding supernatants were collected for IL-2 measurement by ELISA,as illustrated in FIG. 16A. Incubation of Jurkat CD19-aCAR/HLA-A2-iCARwith Raji target cells (‘tumor’) expressing CD19 showed IL-2 secretion,however incubation of these effector cells with Raji-HLA-A2 target cellsexpressing both CD19 and HLA-A2 (‘off-tumor’) resulted in more than 80%inhibition of IL-2 secretion. Conversely, IL-2 secretion was notaffected when CD19 aCAR Jurkat cells were incubated with Raji orRaji-HLA-A2 target cells (FIG. 16B). This result, together with the NFATactivation assay described below points toward the potency of the iCARconstruct to specifically protect normal cells expressing an antigen notexpressed on tumor cells.

Quantitation of Cytokine Release by Flow Cytometry

Transduced T-cells (Jurkat, or primary T-cells) expressing iCAR or aCARor both aCAR and iCAR co-cultured for 6-24 hrs. with recombinant targetcells, expressing iCAR or aCAR or both aCAR and iCAR target antigens ontheir cell surface, will be subjected to Golgi transport blocker (e.g.Brefeldin A, monensin) to enable cytokine intracellular accumulation.T-cells will then be permed and fixed by an inside staining kit (e.g.Miltenyi) and stained with anti CD3 and CD8 and for IL-2 and or INFγ andor TNFα.

Cytokines Secretion Measured by Cytometric Bead Array (CBA) Assay

Cytometric Bead Array (CBA) is used to measure a variety of soluble andintracellular proteins, including cytokines, chemokines and growthfactors.

T-cells (primary T-cells or Jurkat cells) transduced with aCAR or bothaCAR and iCAR constructs (Effector cells) were stimulated with modifiedtarget cells expressing both iCAR and aCAR or aCAR or iCAR targetantigens on their cell surface (FIG. 17A). Following several hours ofco-incubation the effector cells produce and secrete cytokines whichindicate their effector state. The supernatant of the reaction wascollected, and secreted IL-2 was measured and quantified by multiplexCBA assay.

As shown in the FIG. 17B, a specific inhibition of IL-2 secretion wasdemonstrated for aCAR/iCAR transduced Jurkat T-cells co-cultured withtarget cells expressing both target antigens. A decrease of 86% in IL-2secretion was demonstrated when dual CAR (aCAR/iCAR) transduced cellswere co-incubated with target cells expressing both target antigens ascompared to IL-2 secretion resulted from co-incubation of the sameeffector cells with target cells expressing only one target.

NFAT Activation Assay

For determination of T-cell activation as measured by NFAT activation,Jurkat-NFAT cells were transduced with different combinations of aCARand iCAR, as detailed in Table 12. Effector Jurkat-NFAT cell-lines,expressing CD19 aCAR, HLA-A2 iCAR or both, were cocultured with targetcells expressing either CD19 (Raji cells-‘on-target’) both CD19 andHLA-A2 (Raji-HLA-A2 ‘off-tumor’) or HLA-A2 (Thp1 ‘off tumor’) asdescribed in Table 13. As a positive control, effector cells werestimulated in the presence of PMA and Ionomycin, which trigger calciumrelease required for NFAT signaling. Following 16 hrs. incubation at 37°C., luciferase was quantified using BPS Biosciences kit “One stepluciferase assay system” according to the manufacturer's instructions.As expected, Jurkat NFAT cell-line expressing the CD19-CAR constructwere specifically activated in the presence of Raji cell-line expressingCD19, while, no activation was shown when these cells were co-culturedwith Thp1 cell-line which does not express CD19 (FIG. 18).

The inhibitory effect of HLA-A2 iCAR on CD19 aCAR induced NFATactivation can be seen in FIG. 21. Jurkat-NFAT-cell line expressing bothCD19 aCAR and HLA-A2 iCAR was specifically inhibited when co-incubatedwith Raji-HLA-A2, expressing CD19 and HLA-A2 as compared to theactivation induced by Raji cells expressing CD19 only. In contrast,Jurkat-NFAT cell-line expressing only CD19-CAR was similarly activatedby both Raji and Raji-A2 cell-lines. Under these conditions, theinhibition of NFAT activation was calculated as ˜30% (FIG. 19).

The effect of different E/T ratios was tested. Assay was repeatedseveral times with E/T ratios of 10:1, 5:1, 1:1. The results given inFIG. 20 indicate that an increased inhibitory effect can be obtainedwith a higher E/T ratio. The results are presented as a ratio of themean luminescence value from co-culture each effector cell-line with‘off-tumor’ target cells to the mean value from coculture with‘on-target’ presenting cells. As shown, Jurkat-NFAT-cell line expressingboth CD19 aCAR and HLA-A2 iCAR was specifically inhibited whenco-incubated with Raji-HLA-A2 expressing CD19 and HLA-A2 proteins,however, no inhibition was detected when this cell-line was co-culturedwith Raji cell-line expressing CD19 only. On the contrary, Jurkat-NFATcell line expressing CD19 aCAR, was equally activated regardless of theCD19 expressing target cell line it was co-cultured with (Raji orRaji-HLA-A2).

T-Cell Degranulation Assay as Measured by CD107a Staining

Degranulating of T cells can be identified by the surface expression ofCD107a, a lysosomal associated membrane protein (LAMP-1). Surfaceexpression of LAMP-1 has been shown to correlate with CD8 T cellcytotoxicity. This molecule is located on the luminal side of lysosomes.Upon activation, CD107a is transferred to the cell membrane surface ofactivated lymphocytes. CD107a is expressed on the cell surfacetransiently and is rapidly re-internalized via the endocytic pathway.Therefore, CD107a detection is maximized by antibody staining duringcell stimulation and by the addition of monensin (to preventacidification and subsequent degradation of endocytosed CD107a antibodycomplexes).

The transduced T cells will be incubated with the target cells for 6-24hrs in the presence of monensin and will follow CD107a expression on theCD8 T cells by flow cytometry using conjugated antibodies against the Tcell surface markers (CD3,CD8) and a conjugated antibody for CD107a.

Granulation (CD107a) as a marker for the killing potential. The mostcritical function of cytolytic T cells is the ability to kill targetcells. Cytotoxic CD8+ T lymphocytes mediate the killing of target cellsvia two major pathways: perforin-granzyme-mediated activation ofapoptosis and fas-fas ligand-mediated induction of apoptosis. Inductionof these pathways depends on the release of cytolytic granules from theresponding CD8+ T cells. Degranulation is a prerequisite toperforin-granzyme-mediated killing and is required for immediate lyticfunction mediated by responding antigen-specific CD8+ T cells.Cytotoxicity does not require de novo synthesis of proteins by theeffector CD8+ T cell; instead, pre-formed lytic granules located withinthe cytoplasm are released in a polarized fashion toward the targetcell. The lytic granules are membrane-bound secretory lysosomes thatcontain a dense core composed of various proteins, including perforinand granzymes. The granule core is surrounded by a lipid bilayercontaining numerous lysosomal-associated membrane glycoproteins (LAMPs),including CD107a (LAMP-1), CD107b (LAMP-2), and CD63 (LAMP-3). Duringthe process of degranulation, the lytic granule membrane merges with theplasma membrane of the activated CD8+ T cell and the contents of thegranule are then released into the immunological synapse between theCD8+ T cell and the target cell. As a result of this process, thegranular membrane, including CD107a, CD107b, and CD63 glycoproteinstherein, is incorporated into the plasma membrane of the responding CD8+T cell. High-level expression of CD107a and b on the cell surface ofactivated T cells requires degranulation, because degranulationinhibitors, such as colchicine, dramatically reduce cell-surfaceexpression of CD107a and b. Importantly, these proteins are rarely foundon the surface of resting T lymphocytes. Thus, labeling responding cellswith antibodies to CD107a and b and measuring their expression by flowcytometry can directly identify degranulating CD8+ T cells (Betts andKoup, 2004).

Experimental Settings:

PBMC's transduced with iCAR+aCAR/aCAR constructs (Effector cells) arestimulated with either PMA+Ionomycin (Positive Control) or modifiedtarget cells that express iCAR+aCAR/aCAR/iCAR antigens on their cellsurface. During several hours of co-incubation, the effector cellsdegranulate, CD107a can be detected on the cell surface. This expressionis transient and the CD107a is rapidly re-internalized via the endocyticpathway. Therefore, CD107a detection is maximized by antibody stainingduring cell stimulation and by the addition of monensin (to preventacidification and subsequent degradation of endocytosed CD107a antibodycomplexes). BFA is required for optimal cytokine expression.

Example 9. In Vivo Models In Vivo CTL Assay in Human Xenograft MouseModels

To test whether T-cells expressing both aCAR and iCAR constructs coulddiscriminate between the target cells and ‘off-target’ cells within thesame organism and effectively kill the target cells while sparing the‘off-target’ cells will be assessed by an in-vivo CTL assay.

Transduced T-cells with iCAR or aCAR or both iCAR and aCAR will beinjected i.v. to naïve NOD/SCID/γc- or similar mice. Several hourslater, target cells expressing iCAR, aCAR or both will be injected.These targets will be labeled with either CFSE/CPDE or similar celltrace dye in different concentrations (high, medium and low) which willallow further discrimination between them. 18 hrs following targetsinjection, mice will be sacrificed, spleens will be harvested, and theelimination of the specific target will be assessed by FACS. Percentageof specific killing will be calculated according to the formula below:

$\left\{ {1 - \left\lbrack {\left( \frac{\%\mspace{11mu}{{pop}_{high}\left( {{day}\; 1} \right)}}{\%\mspace{11mu}{{pop}_{high}\left( {{day}\; 0} \right)}} \right) \div \left( \frac{\%\mspace{11mu}{{pop}_{medium}\left( {{day}\; 1} \right)}}{\%\mspace{11mu}{{pop}_{medium}\left( {{day}\; 0} \right)}} \right)} \right\rbrack} \right\} \times 100$

Tumor Growth Kinetics in Human Xenograft Mouse Models

NOD/SCID/γc- or similar mice will be inoculated with tumor cells.Inoculation can be i.p/i.v. or s.c. The tumor cells will express eitherthe iCAR target, aCAR target or both. An example for one possible aCARtumor cell line could be the CD19 positive NALM 6 (ATCC, human BALL cellline). Example of tumor cells that express both the aCAR and iCAR (i.e.,‘off-tumor’ cells), is the NALM 6 engineered to express the iCAR epitope(for example HLA-A2) thereby representing the healthy cells. NALM 6 andNALM 6-HLA-A2 can also be engineered to express a reporter gene (e.g.firefly luciferase), for easy detection. Mice will be divided intoseveral study groups inoculated with all possible combinations of targetcells. As an example, one group will be injected with the NALM 6 cellswhile the other will be injected with the NALM-6 expressing the iCARepitope. Several days later, while the tumor has already beenestablished, mice will be infused intravenously with T-cells transducedwith aCAR, or aCAR/iCAR, or iCAR. In addition, control groups ofuntransduced T-cells, no T-cells or T-cells transduced without asignaling domain will also be included. Mice will be monitored untiltumor reaches the experimental end point i.e. the maximal allowed tumorvolume. Monitoring will be by measuring tumor volume by mechanical means(caliper) and also by using in-vivo imaging systems (IVIS). On the endpoint day, mice will be sacrificed, tumor burden will be quantified, andinfiltrating T-cell populations will be analyzed by FACS. To testwhether the T-cells expressing the iCAR construct could discriminatebetween the target cells and ‘off-target’ cells within the sameorganism, we will inject mice with several possible mixtures in severalratios of the ‘on-tumor’/‘off-tumor NALM-6 cells, followed by injectionof transduced T-cells expressing either the aCAR alone or both aCAR andiCAR. Upon sacrifice of the mice the presence of the ‘on-tumor’ and‘off-tumor cells in the spleen and bone marrow will be analyzed by flowcytometry for the two markers, CD19 and the iCAR epitope.

Toxicity and Tumor Growth Kinetics in Transgenic Mouse Models

Transgenic mice that express the human aCAR and iCAR targets will alsobe used to determine the efficacy of the transduced T-cells. Under thesesettings the mice have a fully functional immune system, and thepotential toxicity of the iCAR/aCAR transduced T-cells can be evaluated.The CAR construct will contain scFv that matches the human antigens,while the signaling domains will be modified to activate or inhibitmurine T-cells. One example for such a model is the HHD-HLA-A2 mice thatexpress only human HLA-A2 molecule while all other proteins are solelymurine. The scFv of the CD19 aCAR will be directed in this case to themurine CD19 homolog. Human target cells lacking HLA molecules (e.g. LCL721.221 cells or C1R-neoATCC® CRL-2369™ or similar) will be used. Thetargets will be modified to express the murine CD19. This system willallow monitoring of efficacy and toxicity issues.

mAbs Production

Several pairs of preserved and lost allelic variants identified indifferent tumors are selected and their polypeptide products will servefor the generation of variant specific mAbs using mAb productiontechniques. The discriminatory power of candidate mAbs will be assayedby double staining and flow cytometry experiments orimmunohistochemistry, as determined by binding to recombinant cell-linesexpressing the selected alleles.

All headings and section designations are used for clarity and referencepurposes only and are not to be considered limiting in any way. Forexample, those of skill in the art will appreciate the usefulness ofcombining various aspects from different headings and sections asappropriate according to the spirit and scope of the invention describedherein.

All references cited herein are hereby incorporated by reference hereinin their entireties and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Many modifications and variations of this application can be madewithout departing from its spirit and scope, as will be apparent tothose skilled in the art. The specific embodiments and examplesdescribed herein are offered by way of example only, and the applicationis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which the claims are entitled.

What is claimed:
 1. A method for treating cancer in a subject having a tumor characterized by loss of heterozygosity (LOH), comprising administering to the subject an effector immune cell, wherein the effector immune cell comprises on its cell surface: i) an inhibitory chimeric antigen receptor (iCAR) comprising: an extracellular domain comprising a single chain variable fragment (ScFv) that specifically binds to a single allelic variant of a polymorphic cell surface epitope, wherein the allelic variant is absent from tumor cells of a subject due to loss of heterozygosity (LOH) but present at least on all cells of related normal tissue of the subject; an intracellular domain comprising a signal transduction element that is able to inhibit to effector immune cell, wherein the signal transduction element is homologous to a signal transduction element of PD1, CTLA4, or LIR1; and a hinge domain and a transmembrane domain linking the extracellular domain to the intracellular domain; and ii) an activating chimeric antigen receptor (aCAR) comprising: an extracellular domain that specifically binds to another cell surface antigen, wherein the another cell surface antigen is a tumor-associated antigen or is shared at least by cells of related tumor and normal tissue; an intracellular domain comprising at least one signal transduction element that activates and/or co-stimulates an effector immune cell; and a hinge domain and a transmembrane domain linking the extracellular domain of the aCAR construct to the intracellular domain of the aCAR construct.
 2. The method of claim 1, wherein the signal transduction element is homologous to a signal transduction element of LIR1.
 3. The method of claim 1, wherein the tumor is a solid tumor.
 4. The method of claim 1, wherein the another cell surface antigen is selected from the group consisting of CD19, CD20, CD22, Igκ, ROR1, CD30, CD174, CD33, CD123, NKG2D-L, CD139, BCMA, GD2, FR-α, L1-CAM, ErbB2, EGFRvIII, VEGFR-2, IL-13Rα2, FAP, Mesothelin, c-MET, PSMA, CEA, EGFR, 5T4, GPC3, CD38, CS1, PSCA, CD44v6, CD44v7/8, MUC1, MUC16, PD-L1, IL-11Rα, EphA2, CAIX, and CSPG4.
 5. A nucleic acid molecule encoding an inhibitory chimeric antigen receptor (iCAR) construct, the iCAR construct comprising: i) an extracellular domain comprising a single chain variable fragment (ScFv) that specifically binds to a single allelic variant of a polymorphic cell surface epitope, wherein the allelic variant is absent from tumor cells of a subject due to loss of heterozygosity (LOH) but present at least on all cells of related normal tissue of the subject; ii) an intracellular domain comprising a signal transduction element, wherein the signal transduction element is homologous to a signal transduction element of PD1, CTLA4, or LIR1; and iii) a hinge domain and a transmembrane domain linking the extracellular domain to the intracellular domain.
 6. The nucleic acid molecule of claim 5, wherein the signal transduction element is homologous to a signal transduction element of LIR1.
 7. The nucleic acid molecule of claim 5, wherein the tumor is a solid tumor.
 8. A vector comprising the nucleotide sequence of the nucleic acid molecule of claim 5, wherein at least one control element, such as a promoter, is operably linked to the nucleotide sequence.
 9. The vector of claim 8, further comprising a nucleotide sequence encoding an activating chimeric antigen receptor (aCAR) construct, wherein the aCAR construct comprises: i) an extracellular domain that specifically binds to another cell surface antigen, wherein the another cell surface antigen is a tumor-associated antigen or is shared at least by cells of related tumor and normal tissue; ii) an intracellular domain comprising at least one signal transduction element that activates and/or co-stimulates an effector immune cell; and iii) a hinge domain and a transmembrane domain linking the extracellular domain of the aCAR construct to the intracellular domain of the aCAR construct.
 10. The vector of claim 9, wherein the another cell surface antigen is selected from the group consisting of CD19, CD20, CD22, Igκ, ROR1, CD30, CD174, CD33, CD123, NKG2D-L, CD139, BCMA, GD2, FR-α, L1-CAM, ErbB2, EGFRvIII, VEGFR-2, IL-13Rα2, FAP, Mesothelin, c-MET, PSMA, CEA, EGFR, 5T4, GPC3, CD38, CS1, PSCA, CD44v6, CD44v7/8, MUC1, MUC16, PD-L1, IL-11Rα, EphA2, CAIX, and CSPG4.
 11. An effector immune cell comprising on its cell surface an inhibitory chimeric antigen receptor (iCAR) construct comprising: i) an extracellular domain comprising a single chain variable fragment (ScFv) that specifically binds to a single allelic variant of a polymorphic cell surface epitope, wherein the allelic variant is absent from tumor cells of a subject due to loss of heterozygosity (LOH) but present at least on all cells of related normal tissue of the subject; ii) an intracellular domain comprising a signal transduction element, wherein the signal transduction element is homologous to a signal transduction element of PD1, CTLA4, or LIR1; and iii) a hinge domain and a transmembrane domain linking the extracellular domain to the intracellular domain.
 12. The effector immune cell of claim 11, wherein the signal transduction element is homologous to a signal transduction element of LIR1.
 13. The effector immune cell of claim 11, wherein the tumor is a solid tumor.
 14. The effector immune cell of claim 11, the cell further comprising on its cell surface an aCAR construct comprising: i) an extracellular domain that specifically binds to another cell surface antigen, wherein the another cell surface antigen is a tumor-associated antigen or is shared at least by cells of related tumor and normal tissue; ii) an intracellular domain comprising at least one signal transduction element that activates and/or co-stimulates an effector immune cell; and iii) a hinge domain and a transmembrane domain linking the extracellular domain of the aCAR construct to the intracellular domain of the aCAR construct.
 15. The effector immune cell of claim 14, wherein the another cell surface antigen is selected from the group consisting of CD19, CD20, CD22, Igκ, ROR1, CD30, CD174, CD33, CD123, NKG2D-L, CD139, BCMA, GD2, FR-α, L1-CAM, ErbB2, EGFRvIII, VEGFR-2, IL-13Rα2, FAP, Mesothelin, c-MET, PSMA, CEA, EGFR, 5T4, GPC3, CD38, CS1, PSCA, CD44v6, CD44v7/8, MUC1, MUC16, PD-L1, IL-11Rα, EphA2, CAIX, and CSPG4. 